Repeat-chain for the production of dimer, multimer, multimer complex and super-complex

ABSTRACT

The present invention relates to a method for manufacturing multimers by making repeat-chains comprising repeatedly linked affinity domains binding specifically to monomers, and by using the same to create a repeat-chain/multiple-monomer complex created from the repeat-chains and a multiple number of monomers, thereby facilitating the formation of bond bridges between the monomers in the complex to produce inter-monomeric bond bridged multimer. 
     The present invention relates to a super-complex prepared by cross-binding between repeat-chain/multiple-monomer complexes, and a method for amplifying the effect of monomer through the formation of the said super-complex. Particularly, the repeat-chain/multiple-monomer complex is prepared by containing repeat-chains of binding domain having binding specificity to monomers as active ingredients, and then the super-complex is prepared by cross-binding between such complexes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for manufacturing dimers andmultimers from monomers based on increased yield. To achieve this,multimers in the form of repeat-chain/multiple-monomer complexes areproduced by using repeat-chains of affinity domains specifically bindingto monomers, and multimers made by inter-monomeric bond bridges areproduced by utilizing the fact that inter-monomeric bond bridges areeasily formed between monomers within the complexes. That is, thepresent invention relates to a method for manufacturing large volume ofdimers and multimers by maximizing the formation of multimers in theform of repeat-chain/multiple-monomer complexes by using repeat-chainsas binding matrix, and by increasing the formation efficiency ofproducing inter-monomeric bond bridges among monomers within the formedrepeat-chain/multiple-monomer complexes.

The present invention relates to a super-complex formed by cross-bindingbetween the repeat-chain/multiple-monomer complexes and a method foramplifying the biological and chemical effects of monomers using thesame.

Particularly, repeat-chain/multiple-monomer complexes are produced withcontaining repeat-chains of binding domain having binding specificity tomonomers as active ingredients, and then a super-complex that is anaggregate generated by cross-binding between such complexes is produced.The super-complex contains multiple monomers, so that it can multiplythe biological and chemical effect which is not like any other monomercan give to its target, suggesting the amplification of effects of suchmonomers.

The repeat-chain herein can be prepared in the form of a material or apartial domain of it that has binding affinity to monomers existing inthe nature. There is a wide variety of monomers binding to suchmaterials or domains, which are exemplified by ligands, receptors,antibodies, and enzymes, etc. It is expected that therepeat-chain/multiple-monomer complex and the super-complex generated bythe said complexes can amplify the effects of monomers. Such monomerscan have biological or chemical functional groups linked, conjugated, orfused, and at this time the effect of the functional group might beamplified significantly.

2. Description of the Related Art

Antibody-toxin is produced by fusing toxin to the antibody specificallybinding to cancer cells (Pastan I. et al., Annu Rev Med. 58 (2007)221-237).

Monoclonal antibody (MAb), B3 binds to carbohydrate antigen (LeY)over-expressed on the surface of colon, stomach, ovary, breast, orpulmonary cancer (L. H. Pai, et al., Proc Natl Acad Sci USA 88 (1991)3358-3362; I. Pastan, et al., Cancer Res. 51 (1991) 3781-3787). PE38used for fusion protein of antibody-PE38 is one of the derivatives ofPsudomonas exotoxin (PE) and is the protein toxin having molecularweight 38 kd when N-terminus of PE is truncated (J. Hwang, et al., Cell.48 (1987) 129-136; V. K. Chaudhary, et al., J Biol Chem 265 (1990)16306-16310).

Fab, a fragment having antigen binding part of antibody, includes Fdchain (VH and CH1) and light chain (VL and CL). The Fab used for thefusion protein in the form of Fab antibody-toxin includes Fd chain (VHand CH1) and light chain (VL and CL), too. Fab-toxin has two advantagescompared to single chain Fv-toxin (scFv-toxin, wherein Fv is formed as asingle chain by linking V_(H) and V_(L) with linker and there is noC_(H1) and C_(L)). First, the yield of Fab-toxin is 10 times higher thanthat of scFv-toxin (J. Buchner, et al., Biotechnology (N Y). 9 (1991)157-162; J. Buchner, et al., Biotechnology 10 (1992) 682-685). Second,stability of Fab-toxin is improved in plasma of mouse (M. Choe, et al.,Cancer Res. 54 (1994) 3460-3467).

It has been reported that divalent Fab-toxin dimers ([Fab-toxin]₂) inwhich two Fab-toxin are linked by inter-monomeric disulfide bond bridgeshave higher cytotoxicity than monovalent scFv-toxin (S. H. Choi, et al.,Bull. Kor. Chem. Soc. 22 (2001) 1361-1365; J. H. Park, et al., Mol Cells12 (2001) 398-402; M. H. Yoo, et al., J. Microbiol. Biotechnol. 16(2006) 1097-1103). Disulfide dimers ([Fab-toxin]₂) made byinter-monomeric disulfide bond bridge are manufactured by disulfide bondof intra-monomer counterpartless Cysteine (cys) residues from twoFab-toxin monomers. The intra-monomer counterpartless cysteine residuesare located between Fab domain and the toxin of Fab-toxin monomers.

Dimeric (divalent) antibody-toxin is the simplest form of multimer andexpected to have more advantages than monomeric (monovalent)antibody-toxin. For example, since dimers have two antigen bindingdomains, dimers have stronger binding force than the affinity of eachdomain of monomer and thus, dimers have high avidity for binding. Also,since two of toxin domains (biological or chemical functional group) aretransferred at the same time to the target cells by dimers, it ispossible that antibody-toxin of divalent dimers is able to kill thetarget cells with higher cytotoxicity than monovalent monomers.Furthermore, Fab-toxin has higher stability than scFv-toxin which oftenused to produce recombinant antibody-toxin (D. Rothlisberger, et al., JMol Biol 347 (2005) 773-789).

[B3(Fab)-ext-PE38]₂ in which two B3(Fab)-ext-PE38 monomers are linked byinter-monomeric disulfide bond bridge, can be manufactured by fusing Fabdomains of monoclonal antibody B3 to PE38 (KR 10-0566091), and it isreported that [B3(Fab)-ext-PE38]₂ has 11 times higher cytotoxicity thanB3(Fab)-ext-PE38 when tested on CRL-1739 stomach cancer culture cellline (J. H. Park, et al., Mol Cells 12 (2001) 398-402).

Conventionally, dimers bound by inter-monomeric disulfide bond bridgewere purified after refolding (S. H. Choi, et al., Bull. Kor. Chem. Soc.22 (2001) 1361-1365; J. H. Park, et al., Mol Cells 12 (2001) 398-402; M.H. Yoo, et al., J. Microbiol. Biotechnol. 16 (2006) 1097-1103). Inprevious reported development in connection to refolding process ofdisulfide bonded dimers, the yield of dimers was about 0.014%-0.25%.During the refolding process, these disulfide dimers were formed byrandom accessing of intra-monomer counterpartless cysteine residues oftwo monomers located between Fab domains and PE38. The main producedmolecules of refolding processes were Fab-toxin monomers and the yieldthereof reached to almost 10%. Considering this, although the yields ofdisulfide dimers had been increased through the improvements ofrefolding processes, the yield of dimers were extremely low. Sinceantibody-toxin monomers (Fab-toxin) does not have self-binding-affinityamong themselves, inter-monomeric disulfide bond bridged dimers arecreated through random collisions between the two intra-monomercounterpartless cysteine residues during the refolding process. That is,collision frequency among the intra-monomer counterpartless cysteineresidues of monomers dissolved in refolding buffer solution during therefolding processes is very low and this results the inefficientformation of inter-monomer disulfide bond bridges during the refoldingprocess, and thus the yield of dimmers is extremely low.

As a result, inventors have tried various methods to produceinter-monomer disulfide bonded dimers including methods for producingdisulfide dimers by recycling of oxidation-reduction reactions andchemical cross-linker, but with these methods, the yield of dimers couldnot be greatly improved.

Therefore, in search for a method to produce more dimer, the inventorsconstructed repeat-chains which are recombinant proteins wherein Fabbinding domains of Streptococcal protein G were repeated more than twotimes, and with using this chain as binding matrix, multimers in theform of complexes, in which multiple monomers are bounded simultaneouslyto the chains, were produced. In previous studies, it has been reportedthat the third domain (domain III) of protein G is able to bind to Fabfragment of IgG, and CH1 domain of Fab fragment has high bindingaffinity toward the domains of protein G (J. P. et al., Nature 359(1992) 752-754). As comparing to the refolding process, therepeat-chains of the present invention have the following advantages:High binding affinity for antibody-toxin monomer is shown to the domainsof the repeat-chains. Since the monomers are attached to therepeat-chains and are in fixed state on the repeat-chain, space allowedfor the monomers is quite restricted and monomers cannot move away fromeach other freely, and accordingly, the local concentration of themonomers is very high in the repeat-chain/multiple monomer complexescreated by attaching a plurality of monomers to the repeat-chains.Increased local concentration brings high collision frequency amongintra-monomer counterpartless cysteine residues of each monomerantibody-toxin, and it accelerates the disulfide bond formations betweenmonomers. Therefore, it is possible to produce large quantity ofinter-monomer disulfide bond bridged dimers. Typical example of samemechanism is the proximity effect in enzyme reaction kinetics, whichincreases the local concentration and gives high enzyme reaction rate(Nicholas C. Price and Lewis Stevens. Fundamentals of Enzymology, 3rdEd. Oxford University Press). As a result, the inventors were able toeasily manufacture the antibody-toxin multimer complexes by simplymixing the repeat-chains with monomer antibody-toxins, and completed thepresent invention by confirming that large quantity of[B3(Fab)-ext-PE38]₂ are produced through oxidation and reductionshuffling reactions within the complexes accelerating inter-monomerdisulfide bond bridge formation of cysteine residues between twomonomers.

When a monomer is bound to a target in order to detect the target andthen gives a detection signal, the strength of such detection signalindicating the effect of the monomer is determined by the concentrationof the target material, binding affinity between the target anddetection probe, the concentration of the probe, and the signal effectstrength of the probe. In general, the probe is in the form of amonomeric molecule and the detection target is a binding target of themonomer, and the measured signal is the effect of the monomer.

Immunochromatographic assay, using antibodies, is also called rapidantigen test, lateral flow test particularly when a sample flow islateral, or simply strip test. This assay has been widely used todevelop many diagnostic kits. Immunochromatographic assay has been usedfor the diagnosis of drug abuse, blood components, group A streptococcalantigen, Helicobacter pylori, Mycobacterium tuberculosis, hepatitis Bsurface antigen and antibody, Dengue virus, influenza, and parasite(Plasmodium falciparum for the diagnosis of malaria). The advantages ofusing immunochromatographic assay are simplicity in use, quick detectiontime taking 5˜10 minutes at longest, excellent preservation of reagentsat room temperature, and inexpensive costs, which make the assay thebest and optimum technique for the diagnosis of a variety of diseases.

As a chromogen for immunochromatographic assay, colloidal gold has beenwidely used, and hence colloidal gold-labeled immunoglobulin has beeneffectively used for the diagnosis kit of disease. Colloidal goldparticles conjugated with immunoglobulin have been used for the directdetection of antigen molecules. Leuvering et al developed gold particleagglutination assay which is also called sol particle immunoassay (SPIA)in 1981, and thereafter they developed a pregnancy diagnostic kit usingthe same. Since then, membrane assay using gold has been introduced forthe detection of bacterial, viral, parasitic, and fungal diseases. Inthe meantime, rapid antigen test has been established as rapid fielddiagnostic method characterized by simple and fast detection that can bedone in 10˜15 minutes. Such rapid antigen test is important because itenables early prevention of human contagious disease and early diagnosisand prevention of animal contagious disease as well.

The problem of rapid antigen diagnostic kit being widely used these daysis that the reliable result can only be obtained when the test is donein 2˜3 days from the development of acute disease symptoms. For example,in the case of diagnosing viral disease, if the assay is performed 3days after the symptom is developed, the kit might confirm the resultnegative because virus is rapidly reduced 3 days after the firstexpression. In the case that a patient is young, the decrease of theviral concentration in patient sample starts later, which means it isstill possible to perform the assay even 5 days after the first symptom.However, when a patient is an adult, the viral concentration decreaserapidly with time, so that the detection has to be done at least in 4˜5days after the symptom has been shown. That is, antigen diagnosisdepends on age. The sensitivity of the conventional rapid antigendiagnostic reagents to the sample containing low concentration ofantigen is worse than expected. Rapid diagnostic reagents againstvarious influenza viruses have been introduced in the world market. Thesensitivity of these products has been continuously improved, but thesensitivity against seasonal disease is 60˜83% and the sensitivityagainst new swine virus is only 40-69%. The diagnosis test was performedwith three kits, ‘BinaxNow’, ‘EZ flu A+B’ (Becton Dickinson), and‘Quickvue’ (Quidel), which are available in the US market, in order toconfirm the diagnostic effect thereof. As a result, H1N1 detection rateof BinaxNow was 40% which was the lowest. Quickvue demonstrated 69% H1N1detection rate, while EZ flu A+B showed 49% detection rate (Centers forDisease Control and Prevention (CDC), Evaluation of rapid influenzadiagnostic tests for detection of novel influenza A (H1N1) virus: UnitedStates, 2009. Morb Mortal Wkly Rep 2009; 58:826-829).

The reason of such low sensitivity of the conventional rapid antigendiagnostic reagent is that the virus concentration in the infecteeexcept younger patients is not high enough or the virus concentration inthe sample taken under non-optimum condition for the detection is notenough. That is, if the antigen concentration in the sample taken from apatient having virus infection is not enough, the disease cannot bediagnosed accurately. Therefore, it is requested to establish a methodor a material for the detection of low concentration antigen in asample.

Previously, the present inventors established amultiple-monomer/repeat-chain complex by using antibody-toxin as monomerand repeat-chains of binding domain having binding specificity to Fab ofantibody as a binding matrix (scaffold), and hence the inventorsdeveloped a method to improve the yield of bond bridge multimers byaccelerating bond bridge formation by increasing local concentration ofmonomers and collision frequency among monomers (Korean Patent No.10-1161323). The previous invention of the present inventors related tothe method for mass-production of bond bridge multimers by acceleratingthe formation of bond bridges between monomers by using repeat-chains ofbinding domain having binding specificity to monomers. However, themethod for amplifying the effect of monomer antibody by usingrepeat-chains of binding domain having binding specificity to monomersfor immunoassay has not been reported yet.

The present inventors tried to improve detection sensitivity of lowconcentration antigen diagnosis. As a result, the inventors confirmedthat when repeat-chains of binding domain having binding specificity toantibody monomers were used as signal amplifiers in Western blotting,enzyme-linked immunosorbent assay (ELISA), and RAT (Rapid Antigen Test),it demonstrated high sensitivity at low concentration of antigen.Therefore, the present inventors confirmed that repeat-chains can beeffectively used for the antigen detection analysis using an antibody asa probe monomer by maximizing the effect of the probe antibody monomer.In addition to the antibody-antibody binding protein, there are so manyapplicable pairs of the monomer and the monomer binding molecule in thenature.

SUMMARY OF THE INVENTION

The present invention aims to provide a method for mass-producingmultimers from monomers with inter-monomer bond bridges betweenmonomers, with using repeat-chains of monomer-specific affinity domainsas binding matrix to attach the multiple monomers and to mass-producemultimer complexes in the form of the repeat-chain/multiple-monomercomplexes, and forming the inter-monomer bridge bond between monomers inthe repeat-chain/multiple-monomer complexes, and this provides higherand maximized production methods of multimers linked by inter-monomerbond bridges by the increased collision frequency between theintra-monomer counterpartless cysteine residues in each monomers andsubsequently facilitated formation of the inter-monomer disulfide bondbridges between the monomers compared to the low yield conventionalmethod for producing inter-monomer bridge bonded multimers.

In order to achieve the object explained above, the present inventionprovides a method for preparing multimers, including steps of:

-   -   1) preparing repeat-chains of affinity domains binding        specifically to monomers; and    -   2) preparing repeat-chain/multiple-monomer complexes of the        repeat-chains and the monomers with mixing the repeat-chains of        step 1) and the monomers.        According to the present invention, a method for producing        multimers is provided, which includes steps of:

1) preparing repeat-chains of affinity domains binding specifically tomonomers;

2) preparing repeat-chain/multiple-monomer complexes of therepeat-chains and the monomers with mixing the repeat-chains of step 1)and the monomers; and

3) after forming inter-monomer bond bridges between the monomers withinthe repeat-chain/multiple-monomer complexes of step 2), and separatingrepeat-chains from the complexes in which inter-monomer bond bridges areformed, but not limited thereto.

In an embodiment of this method of dimer formation in whichantibody-toxin monomers are linked by disulfide bond bridges, the yieldof inter-monomer disulfide bond bridge dimers of the antibody-toxinmonomers increased to 200 and more fold of the yield of theconventionally-reported refolding method, and thus the dimer formationyield was increased very high. The dimers produced according to thepresent invention have higher antigen binding strength, highercytotoxicity and higher stability than the previously-reported monomerantibody-toxin, and particularly show approximately 11 times highercytotoxicity to stomach cancer cell line. Therefore, mass production ofthe dimers can be effectively used for the development of cancertreatment agents.

It is another object of the present invention to provide a super-complexformed by cross-binding between repeat-chain/multiple-monomer complexescontaining repeat-chains of binding domain having binding specificity tomonomers such as antibodies, receptors signal transmitters, and enzymes,etc. as active ingredients.

It is also an object of the present invention to provide a method foramplifying the effects of monomers by using the super-complex whichincludes multiple monomers so that the biological and chemical effectson the targets can be increased multiple times.

To achieve the above objects, the present invention provides thefollowing [1]˜[13].

[1] The present invention provides a method for preparing repeat-chainsfor the production of a super-complex, which comprises the step ofpreparing repeat-chains which contain a single or multiple kinds ofmonomer-specific binding domains having at least two binding sites for amonomer repeated therein.

[2] The present invention provides a method for preparingrepeat-chain/multiple-monomer complexes for the production of asuper-complex, which comprises the following steps: 1) preparingrepeat-chains which contain a single or multiple kinds ofmonomer-specific binding domains having at least two binding sites for amonomer repeated therein; and 2) preparing repeat-chain/multiple-monomercomplexes by mixing the repeat-chains of step 1) and the monomers havingat least two binding sites for the repeat-chains.

[3] The present invention provides a method for preparing asuper-complex, which comprises the following steps: 1) preparingrepeat-chains which contain a single or multiple kinds ofmonomer-specific binding domains having at least two binding sites for amonomer repeated therein; 2) preparing repeat-chain/multiple-monomercomplexes by mixing the repeat-chains of step 1) and the monomers havingat least two binding sites for the repeat-chains; and 3) preparingsuper-complexes in forms of aggregates of the complexes by formingcross-binding between the repeat-chain/multiple monomer complexes ofstep 2).

[4] The present invention provides repeat-chains prepared by the methodof the above [1].

[5] The present invention provides repeat-chain/multiple-monomercomplexes prepared by the method of the above [2].

[6] The present invention provides a super-complex prepared by themethod of the above [3].

[7] The present invention provides a method for amplifying the effectsof monomers, which comprises the step of preparing a super-complexbinding to the target of the monomer by mixing the repeat-chain of [4],the repeat-chain-monomer complex of [5], or the super-complex of [6]with the target of the monomer.

[8] The present invention provides the kit for biochemical action,detection, analysis, diagnosis, and treatment which comprises themonomer having at least two binding sites for the repeat-chain andspecificity to a biochemical target or a detection target, and therepeat-chains of binding domain having binding specificity to the saidmonomer.

[9] The present invention provides a method for preparingrepeat-chain-biological and chemical effector group, which comprises thestep of linking, conjugating, or fusing biological and chemical effectorgroup or detection functional group to repeat-chains of binding domainhaving binding specificity to monomers.

[10] The present invention provides a method for preparingmultiple-monomer/repeat-chain-biological and chemical effector groupcomplex, which comprises the following steps: 1) preparingrepeat-chain-biological and chemical effector group by linking,conjugating, or fusing the biological and chemical effector group torepeat-chain of binding domain having binding specificity to monomers;and 2) mixing the monomers to the repeat-chain-biological and chemicaleffector group of step 1).

[11] The present invention provides the repeat-chain-biological andchemical effector group of monomer binding domain prepared by the methodof [9].

[12] The present invention provides themultiple-monomer/repeat-chain-biological and chemical effector groupcomplex prepared by the method of [10].

[13] The present invention provides a method for biological and chemicalaction, detection, analysis, diagnosis, and treatment on the target ofmonomer, which comprises the step of forming a super-complex by mixingthe multiple-monomer/repeat-chain-biological and chemical effector groupcomplex of [12] with the target of monomer.

This invention can be applied to the antibody toxin (immunotoxin)therapeutics, and it makes the curing of the cancer possible bydelivering the super-complexes of antibody-toxin to the cancer targetcell. The super-complex of antibody-toxin has very high curing drugefficacies due to the high multiplicities of monomer antibody-toxin, andit still binds to the target maintaining the same specificity to thetarget. If the binding is one antibody of a super-complex to one antigenthe binding strength is the same, and if the binding is multipleantibodies in one complex to multiple antigen the binding strength ismultiplied to give very strong binding with maintaining the samespecificity

The complexes formed through the association of multiple monomers andrepeat-chain can make insoluble super-complex aggregates bycross-binding between the complexes and precipitates at highconcentration. At low concentration the super-complexes do not have highchance to form bigger super-complex and they do not form precipitatingaggregates. The size of the super-complex has small to large sizedistributions, and the distributions depend on the concentration of themonomers and repeat-chain, and it also depend on the incubation time ofmonomer with repeat chain. At low concentration the small sizesuper-complexes are dominant in population and they are soluble and donot precipitate.

The aggregation and the precipitation, and the size of the super-complexare also dependent on the structure of monomer and repeat-chain. Thenumber of the repeat of the binding domain in the repeat-chaininfluences the chain of cross-binding between the complexes, and thesolubility and molecular size of the monomer also affect the chance ofcross-binding between the complexes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents outline of a method for producing multimers with bondbridges according to the present invention;

FIG. 2 presents the result of the experiments in which the purifiedproteins of the present invention are confirmed with SDS-polyacrylamidegel, in which:

FIG. 2A presents the result of the experiments in which recombinantrepeat-chain proteins of protein G are mixed with SDS-PAGE sample buffersolution, and the mixture is analyzed in reducing 16% SDS-Polyacrylamidegel, wherein the lanes of No. 1 to 7 are TR1˜TR7 respectively (In thisspecification, GR is also used instead of TR to indicate that it is fromprotein G.); and

FIG. 2B presents the result of analyzing B3(Fab)-ext-PE38(Monomer) and[B3(Fab)-ext-PE38]2(Dimer) antibody-toxin molecules, produced accordingto the present invention, in non-reducing 8% SDS-Polyacrylamide gel, inwhich each arrow indicates [B3(Fab)-ext-PE38]2, B3(Fab)-ext-PE38 andB3(Fd)-ext-PE38 from the top (Since (H6-B3(L) has very small molecularweight (i.e., about 25 kDa), this cannot be seen in the drawings); and

FIG. 3 presents the result of analyzing the [B3(Fab)-ext-PE38]2 thatwere formed in the complexes of B3(Fab)-ext-PE38 and TR1˜TR7 proteins(In this specification, GR is also used instead of TR to indicate thatit is from protein G.), by SDS-Polyacrylamide gel, in which:

FIG. 3A presents the result obtained after steps of: binding proteincomplex samples (10 g each) to metal chelating agarose bead (lane 1);reducing the samples with 40 mM of 2-Mercaptoethanol at room temperaturefor 30 min (lane 2); oxidizing the protein complex with 5 mM of oxidizedGlutathione form GSSG, for 2 hours at 37° C. (lane 3); and separating ½of the sample with non-reducing 8% SDS-Polyacrylamide gel, in which theclosed arrows each indicate [B3(Fab)-ext-PE38]2, B3(Fab)-ext-PE38 andB3(Fd)-ext-PE38 from the top) The open arrows indicate TR proteins (Inthis specification, GR is also used instead of TR to indicate that it isfrom protein G.) from complex; and

FIG. 3B presents the result of separating the rest ½ of the sample withreducing 12% SDS-Polyacrylamide gel, in which the two arrows indicateB3(Fd)-ext-PE38 (upper) and H6-B3(L) (bottom). The arrowheads indicateTR proteins (In this specification, GR is also used instead of TR toindicate that it is from protein G.) from complexes (TR1 protein (8 kDaof molecular weight) does not appear in the drawing since the size ofTR1 proteins is too small).

FIG. 4 is the schematic diagrams of expression plasmids constructions ofGR1˜10.

A: Using pGR1 as vector, pGR2 to pGR10 were made. Same method was usedto construct up to pGR20. Each plasmid has one G4S linker between D(III)domains.

B: pGR2-2, 2-3, 2-4 series have two, three, and four G4S linker betweentwo D(III) domains.

FIG. 5 shows the results of SDS-PAGE of purified repeat-chains of domainIII of Protein G. The repeat-chain proteins were analyzed on 16%SDS-PAGE. From lane 1 to 13, the samples are GR1, GR2, GR2-2, GR2-3,GR2-4, GR3, GR4, GR5, GR6, GR7, GR8, GR9, and GR10.

FIG. 6 shows the results of size-exclusion chromatography of the GRcomplexes.

Two vertical arrow indicate the peak of disulfide-dimer (left),[Fab-ext-PE38]2 and monomer (right), Fab-ext-PE38, respectively. Rightpanel shows the comparison of apparent molecular weight of GR complexes.The apparent molecular weight of monomer (▪) and disulfide-bridged dimer() are indicated. The schematic diagram represents the complex ofFab-ext-PE38 with GR3 or GR7.

FIG. 7 shows the results of size-exclusion chromatography of thecomplexes with GR2-2, -3, -4.

A: The size-exclusion chromatography. Two vertical arrows indicate thepeak of [Fab-ext-PE38]2 (left) and Fab-ext-PE38 (right).

B: Elution profiles of size-exclusion chromatography of Fab-ext-PE38associated with GR2-2, -3, and -4. The fractions were electrophoresed innon-reducing 8% polyacrylamide gel. The fractions from 9 to 15.5 ml ofthe elution volume were analyzed. The arrow indicates [Fab-ext-PE38]2.The arrowheads indicate Fab-ext-PE38.

C: The fractions were electrophoresed with reducing 12% polyacrylamidegel. The arrows, arrowheads, and open arrows indicate Fd-ext-PE38, H6-L,and GR proteins, respectively.

FIG. 8 shows the results of analysis on the mixture of [Fab-ext-PE38]2and Fab-ext-PE38 complexed with GR2 or GR3.

A: Size-exclusion chromatography. 395 μg of the mixture of[Fab-ext-PE38]2 and Fab-ext-PE38 was associated with GR2 and GR3proteins. 15 μg of GR protein was used. Vertical arrows and numberindicate peak positions.

B: SDS-PAGE of eluted fractions. The mixture of [Fab-ext-PE38]2 and[Fab-ext-PE38] was used as a control. Non-reducing 8% polyacrylamide gelelectrophoresis was performed. The elution fractions were comparedaccording to the same fraction numbers from 13 to 26, which have theelution volume from 7 to 12.5 ml. The arrow and open arrow indicate[Fab-ext-PE38]2 and Fab-ext-PE38.

FIG. 9 shows the results of comparison of size-exclusion chromatogramsof mixtures of Fab-PE38 monomer and GR2-2, 2-3, 2-4 proteins. Verticalarrows indicates Fab-PE38 monomer (right) and disulfide-dimer (left) asa control. Overlapped chromatograms are associated Fab-monomer withGR2-2 to GR2-4 showing the complex has two Fab-PE38 monomers, dimericform of monomer on repeat-chain.

FIG. 10 shows the results of size-exclusion chromatography for thepurification of the complexes of GR constructs and Fab-PE38 monomer.

A: For the purification of GR1˜6, Hiload superdex-75 pg (26/60) columnwas used.

B: For the purification of GR7˜10, Hiload superdex-200 pg (26/60) columnwas used.

C: For the final purification of Fab-PE38 monomer and disulfide bridgeddimer, Hiload superdex-200 pg (26/60) column was used.

FIG. 11 shows the results of disulfide-bridged-dimer formation ofFab-toxin monomer by redox shuffling in the complex of Fab-toxin monomerwith GR10, GR2-2, GR2-3 and GR2-4.

Lane 1: starting Fab-toxin monomer.

Lane 2: reduction with 40 mM 2-mercaptoethanol for 30 minute at RT.

Lane 3: oxidation with 5 mM glutathione oxidized form (GSSG) for 2 hoursat 37° C. Arrows from top to bottom indicate disulfide-bridged-dimerthat was formed by the redox shuffling reaction, Fab-toxin monomer, andFd chain, respectively.

FIG. 12 shows the result of the amplification of chemiluminescencesignal by GR10 repeat-chain with conventional Western blotting reagents:

Lane A: 20 μg of A431 whole cell lysate (WCL);

Lane 1: 2 μg of A431 WCL;

Lane 2: 1 μg of A431 WCL;

Lane 3: 0.5 μg of A431 WCL; and

GR10 treated Western blot gives 32 fold higher signal than that ofConventional Western blot.

FIG. 13 shows that Chemiluminescence signal of western blot was enhancedby GR10 repeat-chain.

FIG. 14 shows that GR10 significantly enhances the sensitivity of ELISA.

a: 1 g of AGS cell lysate was coated each well. The primary antibody wasserially diluted.

b: The fold increase in A450 was calculated based on the absorbance ofsample of primary antibody alone treated and plotted versus the molarratio used for making the super-complex for each primary antibodydilution factors.

c: With fixed primary antibody dilution factor, 1:120, the AGS celllysate was serially diluted and coated to the 96 well plate.

d: The fold increase in A450 was calculated based on the absorbance ofsample of primary antibody alone treated and plotted versus the molarratio used for making the super-complex for each lysate amount coated onthe plate.

FIG. 15 shows the serial dilution of secondary antibody and signalamplification. It is shown that primary antibody and GR10 complexsignificantly increase the signal of ELISA and is rather consistent inthe tested range of secondary antibody dilution.

FIG. 16 shows the signal amplification in rapid antigen test kit byGR10.

FIG. 17 shows Immuno-fluorescence probing of the human squamouscarcinoma A431 cell with GR1-FITC.

FIG. 18 shows the Cross binding of gold antibody and test line antibodyby GR series.

FIG. 19 shows the Cross binding of gold antibody and test line antibodyby AR, LR, LAR series.

FIG. 20 shows the signal amplification in rapid antigen test kit by GR5

FIG. 21 shows the signal amplification in rapid antigen test kit by GR10

FIG. 22 shows the signal amplification in rapid antigen test kit by GR15

FIG. 23 shows the signal amplification in rapid antigen test kit by GR20

FIG. 24 shows the signal amplification in rapid antigen test kit by AR5

FIG. 25 shows the signal amplification in rapid antigen test kit by LR5

FIG. 26 shows the signal amplification in rapid antigen test kit by LAR3

FIG. 27 shows the result that insoluble precipitation is made by formingsuper-complex between GR10 and IgG. The molar ratios of IgG and GR10were 1:1, 5:1 and 10:1 from left.

FIG. 28 is the SDS-PAGE result that shows that GR10 can makeprecipitation by super-complex much better than GR1 or GR2. 1/36 of eachsample is loaded on a non-reducing 15% SDS-polyacrylamide gel. Lane 1 isthe pellet from GR10+IgG, and lane 2 is the supernatant of GR10+IgG.From lane 3 to 6 are the pellets and supernatants of GR1, GR2 with IgG.Lanes 7 to 10 are the pellet and supernatant of BSA+IgG and IgG only ascontrols.

FIG. 29 shows the precipitations of GR1 to GR10 mixed with IgG. Thecontrols are BSA+IgG and IgG alone. Samples were incubated at roomtemperature and centrifuged at 13000 rpm, 20° C. for 30 minutes. Ininverted micro-centrifuge tubes, precipitate pellets could be observedas white spots. The circles indicate pellets.

FIG. 30 is the SDS-PAGE result that shows precipitation between GR andIgG. From lane 1 to 20 are the pellets and supernatants of GR1˜10 mixedwith IgG. Lanes 21, 22 are the pellet and supernatant of controlBSA+IgG, and lane 23, 24 are those of IgG alone. 1/30 of each sample wasloaded on reducing 15% SDS-polyacrylamide gels.

FIG. 31 shows the result that GR bigger than GR10 also makeprecipitation. On SDS-PAGE 1/10 of each sample was loaded on reducing15% SDS-polyacrylamide gels. Lanes from 1 to 12 are the pellets andsupernatants of GR1, 3, 5, 10, 15, 20 mixed with IgG. The control lane13 and 14 are the pellet and supernatant of IgG alone.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Features and advantages of the present invention will be more clearlyunderstood by the following detailed description of the presentpreferred embodiments by reference to the accompanying drawings. It isfirst noted that terms or words used herein should be construed asmeanings or concepts corresponding with the technical spirit of thepresent invention, based on the principle that the inventor canappropriately define the concepts of the terms to best describe his owninvention. Also, it should be understood that detailed descriptions ofwell-known functions and structures related to the present inventionwill be omitted so as not to unnecessarily obscure the important pointof the present invention.

Hereinafter, the terms used in this invention are described.

The term “binding domain repeat-chain” used in this invention indicatesa recombinant protein or a material produced by repeating the regionhaving binding specificity to monomers such as antibody. The regionhaving binding specificity indicates the binding domain and in the casethat the binding domain is from a natural protein it is a part of theprotein that binds to monomer specifically.

The term “antibody monomer” used in this invention indicates a moleculeoriginated from an antibody, which includes a fragment of an antibodyand molecules fused with other proteins or biological-chemicalfunctional molecules. When this molecule is mixed or contacted withrepeated chains, it is called “antibody monomer”. The natural antibodyhas two heavy chains and two light chains. Considering a set of a heavychain and a light chain as a unit, the natural antibody is a dimer.However, in this invention, when the natural antibody is mixed orcontacted with repeated chains, it is called antibody monomer.

The term “multiple-antibody-monomer/repeat-chain complex” used in thisinvention indicates a complex prepared by mixing or contacting the saidantibody monomer with the repeat-chains of binding domain that bindspecifically to the said antibody monomer.

The term “super-complex of multiple-antibody-monomer/repeat-chaincomplexes” used in this invention indicates a super-complex prepared bycross-binding among the complexes produced by mixing or contacting theantibody monomer with the repeat-chains of binding domain that bindspecifically to the said antibody monomer.

In addition, the term “antigen/multiple-antibody-monomer/repeat-chaincomplex” used in this invention indicates the complex prepared by mixingor contacting the said multiple-antibody-monomer/repeat-chain complexwith antigen.

In addition, the term “super-complex ofantigen/multiple-antibody-monomer/repeat-chain complexes” used in thisinvention indicates the super-complex prepared by mixing or contactingthe super-complex formed by cross-binding among themultiple-antibody-monomer/repeat-chain complexes with antigen or bypreparing mixture of antigen and repeat-chain first and mixing orcontacting the mixture of antigen and repeat-chain with the antibodymonomer.

Hereinafter, the present invention is described in detail.

The present invention relates to a method for manufacturing multimers bymaking repeat-chains comprising repeatedly linked affinity domainsbinding specifically to monomers, and by using the same to create arepeat-chain/multiple-monomer complex created from the repeat-chains anda multiple number of monomers, thereby facilitating the formation ofbond bridges between the monomers in the complex to produceinter-monomeric bond bridged multimer.

The present invention relates to a method for manufacturing multimers bymaking repeat-chain recombinant proteins resulting from the repeatedlinking affinity domain proteins binding specifically to proteinmonomers, and by using the same to create arepeat-chain/multiple-monomer complex created from the repeat-chains anda multiple number of monomers, thereby facilitating the formation ofbond bridges between the monomers in the complex to produceinter-monomeric bond bridged multimer.

More specifically, the present invention relates to a method formanufacturing multimers by making a repeat-chain recombinant proteinsresulting from the repeated linking affinity protein domains bindingspecifically to protein monomers, and by using the same to create arepeat-chain/multiple-monomer complex created from the repeat-chains andmultiple number of monomers, thereby facilitating the formation ofdisulfide bond bridges between the monomers in the complex to produceinter-monomeric disulfide bond bridged multimer.

More specifically, it relates to a method for manufacturing dimers andmultimers by creating repeat-chain recombinant proteins resulting fromthe repeated linking of the domain III (the Fab binding domain) ofStreptococcal protein G, and by using the same as binding matrix(scaffold, skeleton) to create a repeat-chain/multiple-monomer complexcreated from the repeat-chains and multiple number of monomers derivedfrom antibody, thereby facilitating the formation of disulfide bondbridges between the monomers in the complex to produce inter-monomericdisulfide bond bridged multimer.

The method of the present invention can be used to advantage in thelarge-volume manufacture of disulfide bond bridge dimers since it givesup to a 200 fold improvement in the yield of disulfide bond bridgedimers as compared with known refolding methods of the prior art.

The present invention aims to produce large quantity of multimers formedby inter-monomer bond bridges and to provide a method for enhancingyield of bond bridged multimers by using repeat-chains of affinitydomains binding specifically to monomers as binding matrix, andproducing complexes of repeat-chain and multiple numbers of monomers,thereby, increasing local concentration of the monomers on the chains ofthe complexes, increasing frequency of collision between the monomersand facilitating formation of inter-monomeric bond bridges.

While bond bridged multimers are produced by linking multiple monomerswith inter-monomeric bond bridges, the amount of produced bond bridgedmultimers depends on how efficient the formation of bond bridges is. Forthe substance to be used in vivo, the bridge bonds for forming multimersare preferred to be covalent bonds so that the multimers are notdecomposed into monomers and the stability of multimers is maintained invivo. In order for covalent bond bridges to be formed, the contact amongthe bridge bond forming chemical functional groups is required. However,the size of the chemical functional groups in protein is extremelysmaller compared to the total size of protein macromolecule.Accordingly, when the macromolecules are moving freely in reactionsolution, the probability of small chemical functional groups contactingeach other is very low and mass formation of multimers linked withinter-monomer bond bridges is hardly possible unless there is a methodfor greatly increasing the contact of the functional groups.

In order to increase the formation of bond bridges between monomers, thepresent invention creates repeat-chains of affinity domains bindingspecifically to monomers and binds multiple monomers to therepeat-chains making the distance between monomers within severalnanometers, or tens of nanometers or hundreds of nanometers, which isthe scale of molecular size. Therefore, the monomers bound to therepeat-chain cannot freely move away from each other into the solution.That is, the monomers are fixed in the limited space and contact eachother moving in the monomer molecule sized space. Under this condition,local concentration of monomers is extremely high on each repeat-chainto which multiple monomers are bound. Therefore, the frequency ofcontact between bound monomers is remarkably increased and the frequencyof contact between chemical functional groups which can form bondbridges is also increased. Therefore, the formation of multimers isremarkably increased.

The target of the present invention may include not only disulfidecovalent bond bridges linking the proteins, but covalent bonds includingamide bond, ester bond, glycosidic bond, or ether bond, linking themonomers of organic compounds, bio-molecules or proteins, or any othercompounds. The covalent bond bridge may be the most preferable bondbridge to increase stability of multimers, but not limited thereto. Thatis, the concept of the present invention may still be applied when themultimers are formed with bond bridges such as ionic bond. In addition,the concept of the present invention may be applied for improvingformation efficiency of linker connected monomers, as in the case wherethe dimers or multimers are formed by inserting linker chains betweenmonomers to form bridges between monomers producing amonomer-linker-monomer structure (Greg T. Hermanson, BioconjugateTechniques. Academic Press, Inc., 1995; Wong S. S., Chemistry of ProteinConjugation and Cross-Linking. CRC Press, Inc., 1991.)

More specifically, a method according to the present invention toproduce multimers may preferably include the following step 1) to step2) of, but not limited thereto:

1) preparing repeat-chains of affinity domains binding specifically tomonomers; and

2) preparing repeat-chain/multiple-monomer complexes of therepeat-chains and monomers with mixing the repeat-chains of step 1) andmonomers.

Further, the method according to the present invention may additionallyinclude:

3) after forming inter-monomer bond bridges between the monomers withinthe repeat-chain/multiple-monomer complexes of step 2), and separatingrepeat-chains from the complexes in which inter-monomer bond bridges areformed, but not limited thereto.

According to the method, the monomers and the affinity domains bindingspecifically to monomers of step 1) may preferably be originated fromprotein, but not limited thereto. That is, all kinds of molecules havingspecific binding affinity there between may be used as the monomers andthe affinity domains. For example, protein and small organic compoundhaving specific binding affinity thereto may be used as the monomers andthe affinity domains for a method according to the present invention.

According to the method explained above, the preparations ofrepeat-chains of affinity domains binding specifically to monomers(step 1) may include preparing repeat-chains of different kinds ofaffinity domains specifically binding to different kinds of monomerstogether on the same chain and using the repeat-chains as bindingmatrix. Accordingly, hetero-dimer and hetero-multimer may bemanufactured in which different monomers are specifically bound todifferent affinity domains.

According to the method explained above, in step 1), protein monomersand the protein domains having specific binding affinity to monomersthereto may preferably be originated from native form of protein, butnot limited thereto. That is, if monomers of recombinant protein havespecific binding affinity to counterpart affinity domains, therecombinant monomers may be used for producing multimers with bondbridges according to the method of the present invention. DNA fragmentsencoding gene information of the protein may preferably be manufacturedby PCR cloning from chromosomal DNA or from mRNA of organisms expressingthe proteins using forward and reverse pair of primer, but not limitedthereto.

According to the method explained above, the bond bridges in step 3) mayinclude not only the disulfide covalent bond bridges linking theproteins, but also covalent bonds including amide bond, ester bond,glycosidic bond, or ether bond linking the monomers of organiccompounds, bio-molecules or proteins. However, this is not limitedthereto and multimers may be formed by ionic bond.

According to the method explained above, the repeat-chains of step 1)may be prepared by a method including the following a) to c) steps of,but not limited thereto:

a) preparing construct having repeated binding affinity proteins domainsby repeatedly cloning the DNA fragment encoding an affinity domain andan affinity domain connecting linker in an expression vector;

b) preparing transformant by transforming host cell with the constructof step a); and

c) cultivating the transformant of step b), and separating and purifyingexpressed repeat-chain proteins with chromatography, but not limitedthereto.

The construct may preferably have a structure in which the affinitydomain is repeated over more than 3 times within repeat-chains, but notlimited thereto.

Regarding the construct, affinity domain may be linked by linker, andGGGGS may be used as G4S linker, but not limited thereto. The linker maybe extended to, for example, GGGGSGGGGS or GGGGSGGGGSGGGGS, but also notlimited thereto. The G4S linker has highly flexible structure (R. Arai,et al., Protein Eng 14 (2001) 529-532).

According to the present invention, plasmid pTR1 (In this specification,GR is also used instead of TR to indicate that it is from protein G.)was prepared by cutting DNA fragments encoding affinity domain andlinker with NdeI and EcoRI, and cloning the fragments on the vector partof pCW1 cut with the same enzymes.

The DNA fragments encoding affinity domain and G4S linker of pTR1 werecut out again with NdeI and BspEI to separate and purify DNA fragmentsof 226 bp encoding the DNA fragment of affinity domain and one G4S as alinker thereof, and plasmid pTR2 was prepared by cloning the fragmentsonto the large vector fragment obtained by cutting the plasmid pTR1 withNdeI and AgeI. The above-mentioned cloning method was repeated 10 timesand thus the plasmid having the construct in which affinity domains wererepeated 10 times (See Table 1.). The proteins were over-expressed aftertransforming the E. coli BL21 (DE3), and separated and purified withchelating sepharose fast flow chromatography and size-exclusionchromatography.

According to the method, the monomers of step 2) have matrix bindingsequence (MBS), the site specifically binding to the affinity domainbinding matrix. When the monomers have an binding target, monomers havetarget binding sequence (TBS) for the target and the target substance ofmonomers may be used to prepare affinity domain repeat-chains, in thiscase, target binding sequence (TBS) and matrix binding sequence (MBS) ofmonomers are the same one. Also, when affinity domain repeat-chains areprepared by non-target substance of monomers which is specificallybinding to monomers, but not to target binding sequence (TBS) of themonomers and used to make binding matrix, the matrix binding sequence(MBS) of monomers is different from the target binding sequence (TBS)and the monomers have matrix binding sequence (MBS) and target bindingsequence (TBS) separately.

Regarding the formation of repeat-chains-multiple monomers complex ofstep 2), it is possible to manufacture hetero-dimer and hetero-multimermade of different monomers each specifically binding to each differentaffinity domains by using repeat-chains of affinity domains havingdifferent binding specificity on the same chain.

According to the method, inter-monomer counterpartless cysteine ispreferably necessary to manufacture disulfide bond bridged multimersfrom a complex of multiple monomers of step 2), but not limited thereto.That is, various kinds of chemical bonds and chemical cross-linker maybe used for bond bridges between monomers as well as disulfide bondbridges between the counterpartless cysteines on monomers. If themonomer has any counterpartless cysteine for bond bridge, onecounterpartless cysteine is preferably included therein, but not limitedthereto.

According to the method, the monomers of step 2) may be used to make newkind of monomers by linking the monomers to functional proteins (i.e.,functional group, F) with extension sequence (Ext).

According to the method, functional group (F) may include all ofbiological and chemical compound such as enzyme, toxin functional groupprotein or virus, pharmaceutical compound for drug activity, functionalgroup (F) such as liposomes, bio-sensor or prodrug.

The extension sequence (Ext) links monomers and functional groupproteins (F) to fuse monomers and functional group proteins and theextension sequence (Ext) may include counterpartless cysteine onmonomers, but not limited thereto. The counterpartless cysteine isoxidized between the two monomers and forms disulfide bond bridges;therefore, covalently bonded dimers are formed. Also the extensionsequence (Ext) includes flexible amino-acid sequence (Flx) between thelast one of counterpartless cysteines on monomers of disulfide bondbridge dimer formation and hetero functional groups (F). The flexiblesequence (Flx) is preferably comprised with sequence including GASQENDamino-acids (i.e., glycine, alanine, serine, glutamine, glutamic acid,asparagines and aspartic acid) which is not bulky amino-acid, but notlimited thereto.

According to the method, the mixing of step 2) is preferably performedby mixing with the monomers and incubating 3˜5° C. overnight andincubating at 35˜40° C. for 1˜2 hrs. It is more preferable to perform byincubating 4° C. overnight and incubating for 1 hr at 37° C., but notlimited thereto.

According to the method, the inter-monomeric bond bridges of step 3)include not only the disulfide covalent bond bridges with proteinmonomers, but covalent bonds including amide bond, ester bond,glycosidic bond, or ether bond, linked to monomers of organic compounds,bio-molecules or proteins. The covalent bond bridge may be the mostpreferable bond bridge to increase stability of multimers, but notlimited thereto. That is, the concept of the present invention may stillbe applied when the multimers are formed with bond bridges such as ionicbond.

According to the method, regarding the formation of bond bridges, bondbridge-heteromultimer may be manufactured by forming bond bridgesbetween different monomers in a complex of hetero-multiple monomersformed by repeat-chains of affinity domains having different bindingspecificities on same chain.

According to the method, bond bridges of step 3) may be formed by usingthe various existing methods (Greg T. Hermanson, BioconjugateTechniques. Academic Press, Inc., 1995; Wong S. S., Chemistry of ProteinConjugation and Cross-Linking. CRC Press, Inc., 1991.). If disulfidebond bridges are used, the thiol group (—SH) of counterpartless cysteineon monomers is reduced first to be in the —SH form. This reduction ispreferably performed by adding the protein complex into reduction buffersolution including 2-merchaptoethanol, but not limited thereto. That is,regarding the reduction, the added amount of 2-merchaptoethanol ispreferably 20-40 mM for full reduction of cysteine residues of monomers,but not limited thereto.

According to the method, if disulfide bond bridges are used to form bondbridges of step 3), multimers having disulfide bond bridges are producedby the oxidation of reduced cysteine residues of each of the pairedmonomer. The oxidation between the monomers is preferably performed byadding reduced protein complex into oxidation buffer solution containingglutathione oxidized form (GSSG), but not limited thereto.

In the present invention, the reduction of protein complex is preferablyperformed by adding Tris-HCl (pH8.2) in which 2-merchaptoethanol iscontained, at room temperature, but not limited thereto. The reducedprotein complex is preferably washed with the buffer solution includingMOPS (pH6.5), but not limited thereto. The complex is washed 3 timesmore with Tris-HCl (pH8.2); oxidation buffer solution including GSSG andTris-HCl (pH8.2) is added thereto for oxidation; and this is incubatedat 37° C. for 2 hrs. According to the method, the temperature and timefor incubation is preferably 37° C. and for 2 hrs respectively, but notlimited thereto.

According to the method, the analysis of bond bridge multimers of step3) is preferably performed with chromatography, but not limited thereto.All methods which are commonly used for separation and purification ofproteins may be used.

According to the method, multimers of step 3) are preferably the dimersin which monomers are linked by disulfide bonds, but not limitedthereto. The monomers according to the present invention can be offusion protein molecule in the form of ‘binding domain (B)-extensionsequence (Ext)-functional protein (F), (B-Ext-F)’, wherein targetbinding sequence (TBS) and independent matrix binding sequence areexisted in the binding domain (B). In case of the extension sequenceincludes counterpartless cysteine in monomers, disulfide bond bridgesare formed between the counterpartless cysteine in monomers of twobinding domains (B)-extension sequence (Ext)-functional protein (F)molecules; therefore, the dimers are formed in the form of [bindingdomain (B)-extension sequence (Ext)-functional proteins (F),(B-Ext-F)]₂.

In order to complete the present invention, the inventors usedantibody-toxin having Fab protein fragment of antibody as monomer'starget binding domain (B) as in the form of ‘binding domain(B)-extension sequence (Ext)-funtional protein (F)’ monomer and domainIII (DIII), Streptococcal protein G's Fab binding domain, as affinitydomain having specific binding affinity to Fab domain of antibody-toxin.All kinds of the antibody molecules (antibody derived molecule monomersor antibody-functional group fusion molecule monomers) that areoriginated from antibody and contains Fab fragment of antibody as itspart can be used as monomers as well as the ones in antibody-toxin form.In order to maximize the formation of dimers having disulfide bondbridges between monomers ([antibody derived molecule monomer]₂),recombinant repeat-chains proteins are prepared wherein Fab bindingdomains (i.e., domain III) of Streptococcal protein G are repeated over2 times, and the prepared proteins are used as binding matrix forpreparing [antibody derived molecule monomer]₂. More specifically, therepeat-chains and antibody derived molecule monomer proteins are mixedto manufacture the multimers in the form of complex of recombinantrepeat-chains-antibody derived molecule multiple monomers, in which manyantibody monomers are bound to one recombinant repeat-chains proteinwith non-covalent bonds, and the cysteine residues of antibody monomersare reduced and oxidized to form the disulfide bond of cysteine residueslocated between Fab fragment and toxin of antibody derived moleculemonomer, thus [antibody derived molecule monomers]₂ is prepared.

For comparing the formation level of [antibody derived moleculemonomers]2 based on the present invention and the one of based onprevious refolding method, the inventors of the present invention usedSDS-PAGE and analyzed the yield of [antibody derived molecule monomers]2through measuring the SDS-PAGE band density, and the result was that theyield of [antibody derived molecule monomers]2 based on the presentinvention is remarkably higher than the reported one performed based onthe refolding method. That is, the yield of [antibody derived moleculemonomers]₂ is in 36 to 52% range of the total antibody derived moleculemonomers binding to recombinant repeat-chain proteins wherein Fabbinding domain (i.e., domain III) of Streptococcal protein G is repeatedover more than 2 times. Accordingly, it is confirmed that the yieldrange is about 200 and more times higher than the reported yield ofexisting refolding method.

As a result, it is confirmed that disulfide bond bridge dimers frombound antibody derived molecule monomers may be formed usingrepeat-chains of Fab binding domain (i.e., domain III) of protein G, andthe use of the repeat-chains increases the formation of [antibodyderived molecule monomers]₂ drastically. By using repeat-chains of Fabbinding domains (i.e., domain III) of protein G and its bindingspecificity to antibody molecule monomers, simple reduction andoxidation can produce large amount of [antibody derived moleculemonomers]₂.

In the present invention, the repeat-chains having specific bindingaffinity are characterized to form a repeat-chain/multiple-monomercomplex. Therefore, as fixing the repeat-chains on resin, it is possibleto perform affinity purification with high efficiency for thepurification of useful protein molecules in monomeric or multimeric formin biotechnology and medical industry (Zachariou M., AffinityChromatography: Methods and Protocols (Methods in Molecular Biology)Humana Press; 2nd edition).

In addition, the present invention provides the gene construct encodingthe expression of recombinant repeat-chain protein of affinity domainsbinding specifically to monomers.

The present invention prepared the gene construct encoding recombinantrepeat-chain proteins of domain III (Fab binding domain) of Protein G.

The recombinant repeat-chain proteins preferably include one linker, G4S(GGGGS), between two of Fab binding domains, but not limited thereto.

The recombinant repeat-chain proteins preferably have the structure inwhich domain III (Fab binding domains) of protein G is repeated 3 to 10times, but not limited thereto.

Also, the present invention provides expression vector including geneconstruct.

The expression vector is preferably connected to the desired gene of thespecific nucleic acid sequencing having information for controlling genetranscription toward mRNA and translation to proteins to give goodexpression of desired protein and to increase the possibility of proteinformation.

Furthermore, the present invention provides transformant produced by theexpression vector in the host cells.

The inducing of the expression vector into host cells is performed byone of appropriate methods including transformation, transfection,conjugation, protoplast fusion, electroporation, calciumphosphate-precipitation or direct microinjection. The host cells may beprokaryotic or eukaryotic cells. Eukaryotic cells are preferable.Eukaryotic cells are the cells of mammals such as CHO cells. These cellsare preferably the cell providing correct folding at correct position ortransformation for protein molecule including glycosylation aftergenerating the proteins, but not limited thereto.

The present invention confirmed that [antibody derived moleculemonomers]2 is able to be produced in large quantity from antibodyderived molecule monomers with using recombinant repeat-chain proteinsas binding matrix wherein Fab binding domain (i.e., domain III) ofStreptococcal protein G is repeated 3 to 10 times within the recombinantrepeat-chain proteins.

Accordingly, the repeat-chains of affinity domains having specificbinding affinity may be used to improve the formation of dimers andmultimers between monomers. Also, gene construct encoding theserepeat-chains, expression vector including the gene construct andtransformant transformed with the expression vector are very useful formass formation of bond bridged multimers between antibody derivedmolecule monomers.

The present invention provides a method for preparing repeat-chains,which comprises the step of preparing repeat-chains which contain asingle or multiple kinds of monomer-specific binding domains having atleast two binding sites for a monomer repeated therein.

The present invention also provides a method for preparingrepeat-chain/multiple-monomer complexes, which comprises the step ofpreparing repeat-chain/multiple-monomer complexes by mixing therepeat-chains prepared by the above method with multiple monomers.

The present invention also provides a method for preparing asuper-complex, which comprises the step of preparing an aggregate of thecomplexes by cross-binding among the repeat-chain/multiple-monomercomplexes prepared by the above method.

In the above method, the said monomer is preferably a protein, which ismore preferably selected from the group consisting of antibodies,ligands, receptors or fragments thereof, or recombinants thereof, orderivatives thereof, or fusions of biological or chemical functionalgroup therewith.

In the above method, the antibody is preferably selected from the groupconsisting of fragments of an antibody, Fab fragment, fragmentscontaining Fab fragment, Fv fragment, fragments containing Fv fragment,Fc fragment, and fragments containing Fc fragment.

In the above method, the binding domain is preferably a protein, andmore preferably a microorganism derived protein, and most preferablyselected from the group consisting of Streptococcal protein G,Staphylococcus aureus protein A, Peptostreptococcus magnus protein L,and derivatives thereof.

The repeat-chain of the present invention can include flexible linkerchain that helps free rotation of each domain in between repeatedbinding domains and maintains distance between domains. This preventsbinding reaction rate constant and binding reaction equilibrium constantfrom being reduced because of inter-monomer collision steric hindranceduring monomer binding, resulting in the improved binding reactionequilibrium.

The repeat-chain of the binding domain facilitates free rotation ofbinding domain in the chain and bending of chain as well with the aid ofthe flexible. Therefore, in the complex formed by mixing repeat-chainswith monomers, each binding monomer can have satisfactory rotationalfreedom and vibrational freedom (bending freedom). That is, the bindingmonomer has little limitations in the direction of rotation and anglesfor bending but rotates freely in an enough range of directions andangles to avoid inter-monomer collision. Therefore, it is possible formultiple monomers to bind repeat-chain simultaneously. The flexiblelinker sequence makes the room between binding domains in repeat-chainlarge enough so as not to hinder the approaching monomers each other,resulting in the successful simultaneous binding to the repeat-chain.The natural protein that has binding domain usable for repeat-chains ofthe present invention usually does not have such flexibility in itsstructures of binding domains. Natural proteins do not allow suchfreedom in around their binding domains, so that they cannot providehigh level rotation freedom and vibrational freedom to the monomers.

The binding domain used in the repeat-chain of the present invention ispreferably a fragment of a natural protein molecule, and this fragmenthas lower molecular weight than a whole natural protein. Makingartificial repeat-chain by using such low molecular weight bindingdomain is advantageous in the construction of repeat-chain that has lowmolecular weight but has many binding domains, compared with a highmolecular weight natural protein molecule. Therefore, multiple monomerscan possibly bind to the repeat-chain of low molecular weight. Therepeat-chain that has multiple binding domains but has low molecularweight is easy to produce and purify, which is a big advantage thatnatural protein does not have. In this invention, the repeat-chain thatcontains up to 20 repeats of binding domain has been constructed, butthis invention is not limited thereto.

When the repeat-chain having multiple binding domains but low molecularweight is used, the ratio of monomer to binding molecule (naturalmicroorganism protein molecule, or the repeat-chain of the presentinvention) is increased, compared with natural molecule, suggesting thatthe effect of monomer can be amplified significantly per bindingmolecule. The repeat-chain herein has low molecular weight, so that itcan be effectively produced as an artificial protein.

In this invention, when monomer has sites a′ and b′ therein and bindingdomain has binding sites for them, a and b, the repeat-chain has to havea and b and the monomer has to have at least one of each a′ and b′ toform a super-complex of multiple-monomer/repeat-chain complexes bycross-binding between complexes.

In this invention, the monomer is in the form of (a′ b′), indicatingthat a′ and b′ exist together in one monomer. In the binding domain ofrepeat-chain, the binding sites a and b can be together in one bindingdomain to form the repeat-chain having the form of(ab)-(ab)-(ab)----(ab) or the binding sites a and b can be separately indifferent binding domains to form the repeat-chain having the domain aand domain b repeats in the form of a-b-a-b-a-b----a-b. In that case,the number or the order of the independent (separate) domain a and b isnot limited. In addition, when a=b, the monomer is (a′ a′) and therepeat-chain is (aa)-(aa)-(aa)--- or a-a-a-a-a-a------.

The repeat-chain for the formation of cross-binding between complexesthat can make super-complex can be made to have the binding domain c andd that bind to each other and are independent from the binding domainfor the multiple-monomer/repeat-chain complex formation. If the chain isconstructed to be c-a-a-a-------d, where the monomer binds to domain a,the cross-bindings between the complexes are formed through the bindingof c to d, c-a-a-a-----a-d . . . c-a-a-a-----a-d . . . c-a-a-a-----a-d .. . , where . . . indicates the cross-bindings between the repeat-chain,and the super-complexes are formed to give amplification of monomer thatare bound to these repeat-chains. The structure of repeat-chain shouldbe rigid enough for the c and d domains in one repeat-chain not to bindto each other. If c and d domains bind each other in one chain thepossibility of forming super-complex is very low because there is verylow chance for cross-binding between complexes ofmultiple-monomers/repeat-chain. It is also needed for the repeat-chainnot forming c of one chain to d of other chain binding between thechains before mixing with the monomers. If it forms chain to chainbinding it is very difficult to handle the molecules and to makecomplexes with monomers.

The present invention also provides repeat-chains which contain a singleor multiple kinds of monomer-specific binding domains having at leasttwo binding sites for a monomer repeated therein.

The present invention also provides repeat-chain/multiple-monomercomplexes prepared by mixing multiple monomers to the said repeat-chain.

The present invention also provides a super-complex which is theaggregate of the said complexes generated by cross-binding among therepeat-chain/multiple-monomer complexes.

The said monomer is preferably a protein, which is more preferablyselected from the group consisting of antibodies, ligands, receptors orfragments thereof, or recombinants thereof, or derivatives thereof, orfusions of biological or chemical functional group therewith.

The antibody is preferably selected from the group consisting offragments of an antibody, Fab fragment, fragments containing Fabfragment, Fv fragment, fragments containing Fv fragment, Fc fragment,and fragments containing Fc fragment.

The binding domain is preferably a protein, and more preferably amicroorganism derived protein, and most preferably selected from thegroup consisting of streptococcal protein G, Staphylococcus aureusprotein A, Peptostreptococcus magnus protein L, and derivatives thereof.

The present invention also provides a method for amplifying the effectof monomer, comprising the step of preparing a super-complex by mixingthe repeat-chain, the repeat-chain/multiple-monomer complex, or thesuper-complex thereof to the target of the monomer.

In the above method, the step of measuring the effect of the monomer onthe target of the monomer can be additionally included.

In the above method, the target of the monomer is preferably selectedfrom the group consisting of antigens, antibodies, peptides, proteins,bacteria, viruses, fungi, and the fragments thereof but not alwayslimited thereto.

The bacteria herein are preferably selected from the group consisting ofHelicobacter pylori, Mycobacterium tuberculosis, and Chlamydiatrachomatis, but not always limited thereto.

The virus herein is preferably selected from the group consisting ofinfluenza, foot and mouth disease virus, human papilloma virus (HPV),Dengue fever virus, hepatitis C virus, and hepatitis B surface antigenand antibody, but not always limited thereto.

In this invention, the measurement of the effect of the monomer ispreferably performed by using monomer-marker conjugate or secondaryprobe (antibody)-marker conjugate, and labeling substrate of the marker,but not always limited thereto.

The super-complex of the present invention gives the opportunity toamplify the effect of the monomer, for example the signal amplification,etc, since it contains multiple monomers suggesting that it bearsmultiple biological and chemical effects on the target of the monomer,compared with a single monomer.

The present invention also provides an analysis kit containing themultiple monomers having analysis target specificity and at least twobinding sites to the repeat-chain in one monomer and the repeat-chainsof binding domain having binding specificity to the said monomers.

The said kit is preferably composed of,

1) repeat-chains of binding domain having binding specificity tomonomers;

2) monomers binding specifically to the analysis target;

3) secondary probe conjugate labeled with a marker showing labelfunction through the reaction with substrate;

4) marker substrate solution for the reaction with the said marker;

5) washing buffer to be used in each reaction stage; and

6) markering reaction stop buffer, but not always limited thereto.

The kit facilitates the analysis selected from the group consisting ofimmunohistochemical techniques, immunoblot, immunoprecipitation, enzymelinked immunosorbent assay (ELISA), agglutination, immunochromatographicassay, and radio-immuno assay.

The said marker is preferably selected from the group consisting ofhorseradish peroxidase (HRP), alkaline phosphatase, colloid gold,fluorescein, Quantum dot, glucose oxidase, luciferase,beta-D-galactosidase, malate dehydrogenase (MDH), acetylcholinesterase,radio-isotope, and dye, but not always limited thereto.

The chromogenic substrate herein is preferably selected from the groupconsisting of 3,3′,5,5′-tetramethyl bezidine (TMB),2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), diaminobenzidine (DAB),3-amino-9-ethylcarbasole, 5-bromo-4-chloro-3-indolylphosphate/iodonitrotetrazolium (BCIP/INT), new fuchin (NF), and fast redTR salts, but not always limited thereto.

The present invention also provides a method for preparingrepeat-chain-detection functional group which has detection functionalgroup added to repeat-chain, containing the step of linking,conjugating, or fusing detection functional group to repeat-chains ofmonomer-specific binding domains.

The present invention also provides a method for preparingmultiple-monomer/repeat-chain-detection functional group complex,comprising the following steps:

1) preparing repeat-chain-detection functional group by linking,conjugating, or fusing detection functional group to repeat-chains ofmonomer-specific binding domains; and

2) mixing monomers to the repeat-chain-detection functional groupprepared in step 1).

The present invention also provides repeat-chain-detection functionalgroup of monomer binding domain prepared by linking, conjugating, orfusing detection functional group to repeat-chains of monomer-specificbinding domains.

The present invention also providesmultiple-monomer/repeat-chain-detection functional group complex whichis prepared by mixing multiple monomers to the repeat-chain-detectionfunctional group of monomer binding domain.

The present invention also provides a method for detecting the target ofthe monomer, containing the step of forming a super-complex bound to thetarget of the monomer by mixing themultiple-monomer/repeat-chain-detection functional group complex to thetarget of the monomer.

In the above method, the step of measuring the detection level ofmonomer for the target of the monomer can be additionally included.

In the above method, the detection functional group is preferablyselected from the group consisting of Cy-3, Cy-5, FITC, GFP (greenfluorescent protein), RFP (red fluorescent protein), and Texas Red, butnot always limited thereto.

In the above method, if the monomer is an antibody, the antibody ispreferably selected from the group consisting of fragments of anantibody, Fab fragment, fragments containing Fab fragment, Fv fragment,fragments containing Fv fragment, Fc fragment, and fragments containingFc fragment.

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Preparation of Repeat-Chains Construct of Fab Binding Domainfrom Streptococcal Protein G

The inventors obtained domain III of protein G from Korean Collectionfor Type Cultures (KCTC) and performed PCR cloning with P1[5′-GGGCATATGC ATCAC CATCA CCATC ACACC GGTAC ACCAG CCGTG ACAA-3′(SEQ ID No: 1)]and P2[5′-CCCGA ATTCT TATCC GGACC CGCCT CCACC TTCAG TTACC GTAAA-3′(SEQID No:2)] primers from chromosomal DNA of Streptococcus. The PCRproducts (243 bp) were cut with NdeI and EcoRI, and was cloned intovector pCW1 which was cut with the same. The encoding sequence of thedomain III was confirmed by dideoxy-DNA sequencing. G4S linker for eachdomain III was added as spacer and thus, pTR1 was obtained (In thisspecification, GR is also used instead of TR to indicate that it is fromprotein G.). The plasmid pTR1 was cut again with NdeI and BspEI and 225bp fragment encoding domain III and one of G4S was ligated to the largefragment of identical pTR1 plasmid cut with NdeI and AgeI, thus pTR2encoding the two time Tandem Repeat of domain III was obtained. Theplasmid pTR2 was cut with NdeI and AgeI again, and the large fragmentwas ligated to 226 bp fragment to produce new plasmid ‘pTR3’. Using thecloning explained above, plasmid (pTR10) having up to 10 repeats ofdomain III of protein G was prepared (See Table 1). Based on theexisting method, pMC75H encoding H6-B3(L) was prepared (See KR10-0566091).

TABLE 1 Used plasmids and proteins in the present invention PlasmidProteins References pCW1 B3(Fd)-ext-PE38:Fd- J. H. Park, et al.,SKPSIST- Mol Cells 12(2001) KASG₄C(G₄S)₂GGPE- 398-402 PE38^(a) pMC75HH6-B3(L): (His)₆ ^(b)- The detailed Light chain description of thepresent invention pTR1~10 TR1~10: (His)₆- The detailed (DIII-G₄S)_(n), n= 1~10^(c) description of the present invention ^(a)SKPSIST: a mutatedsequence of natural-type hinge sequence(CKPCICT); ext:SKPSIST-KASG4C(G4S)2GGPE: extended peptide chains having cysteineresidues (Cys residue); G4S: amino acid sequence of GGGGS; PE38:truncated Pseudomonas Exotoxin of 38 kd; ^(b)(His)6: 6 of Histidinetags; and ^(c)DIII: domain III of Streptococcal protein G.

Based on the existing method, the repeat-chains were over-expressed (J.H. Park, et al., Mol Cells 12 (2001) 398-402).

Pure lysate was separated with chelating sepharose fast flowchromatography (Amersham Bioscience, Sweden) and performedsize-exclusion chromatography with Hiload Superdex-75 pg or HiloadSuperdex-200 pg(26/60 (Amersham Bioscience, Sweden). Proteins of all theconstructs were purified over 95% purity (See FIG. 2A).

Example 2 Preparation of B3(Fab)-ext-PE38 and [B3(Fab)-ext-PE38]₂ byRefolding

Based on the existing method, the inventors performed over-expression,preparation and refolding of inclusion body of B3(Fd)-ext-PE38 andH6-B3(L) (J. H. Park, et al., Mol Cells 12 (2001) 398-402). The refoldedproteins were purified with Q-sepharose FF, Hitrap protein G HP, andHiload Superdex-200 pg(26/60)(Amersham Bioscience, Sweden)chromatography.

More specifically, B3(Fab)-ext-PE38 was prepared by inter-chaindisulfide bond between B3(Fd)-ext-PE38 and H6-B3(L). The two cysteineresidues related to inter-chain disulfide bond located one on the end ofCH1 domain of B3(Fd)-ext-PE38 and other on the end of CL domain ofH6-B3(L). Also, during the refolding process, [B3(Fab)-ext-PE38]2 wasalso produced by inter-monomeric disulfide bond between two monomers bythe intra-monomer counterpartless cysteine residues located on the extposition of B3(Fab)-ext-PE38 monomer. After performing size-exclusionchromatography, 95% purity of B3(Fab)-ext-PE38 and [B3(Fab)-ext-PE38]₂was measured as a result of conducting densitometric analysis ofnon-reducing SDS-polyacrylamide gel (See FIG. 2B). The conventionalrefolding yield of [B3(Fab)-ext-PE38]₂ was about 0.06%, while the methodof the present invention provided 200 times increased yields.

Example 3 Binding of B3(Fab)-ext-PE38 Monomers with Repeat-ChainsConstruct from Protein G Domain III

For the binding reaction between purified repeat-chain construct ofprotein G domain III and monomer B3(Fab)-ext-PE38, the inventors mixedB3(Fab)-ext-PE38 (715 μg) and TR proteins (28 μg each) (In thisspecification, GR is also used instead of TR to indicate that it is fromprotein G.), warmed the mixture at 4° C. over-night, and then warmed themixture at 37° C. for 1 hr. The reacting mixture was separated bysize-exclusion chromatography (Superdex-200TM HR). The elusion profileof the protein complex was compared to those of B3(Fab)-ext-PE38 only(Kav=0.33) or [B3(Fab)-ext-PE38]2 only (Kav=0.20) as controls. The Kavvalue of the eluted protein peak was calculated with [Formula 1].

$\begin{matrix}{{Kav} = \frac{\left( {{Ve} - {Vo}} \right)}{\left( {{Vt} - {Vo}} \right)}} & \left\lbrack {{Formula}\mspace{14mu} 1} \right\rbrack\end{matrix}$

wherein Ve is elution volume of the peak, Vo is void volume of thecolumn, which is the elution volume of blue dextran 2000; and Vt is bedvolume of superdex-200 column.

B3(Fab)-ext-PE38(Kav=0.22) bound to TR3 gave similar elution volume tothat of [B3(Fab)-ext-PE38]2. Other complexes had 2 or more ofB3(Fab)-ext-PE38 monomer molecules bound simultaneously to TR chainsexcept TR2. TR3 to TR6 complexes were dimers of B3(Fab)-ext-PE38 monomerand TR7˜10 complexes were trimers.

Example 4 Preparation of [B3(Fab)-ext-PE38]₂ Dimer from the Complexes ofRepeat-Chains of Protein G Domain III and B3(Fab)-ext-PE38 Monomers

In order to produce [B3(Fab)-ext-PE38]₂ dimer in the complexes of thepurified TR1˜TR10 chain proteins (In this specification, GR is also usedinstead of TR to indicate that it is from protein G.) andB3(Fab)-ext-PE38 monomers bound thereto, the inventors performedreduction with 2-Mercaptoethanol, oxidation reactions to the boundmonomer molecules on the chains with oxidized form of glutathione(GSSG), and the products were analyzed by non-reducing (FIG. 2A) andreducing (FIG. 2B) SDS-PAGE.

More specifically, The protein complex (10 μg) was mixed withmetal-chelating Sepharose beads (20 μl) in a microtube for 1 h at 10° C.The metal-chelating Sepharose beads were in a 50% suspensionequilibrated with 100 mM Tris-HCl buffer (pH 8.2). The immobilizedprotein complex was reduced by the addition of 40 mM 2-merchaptoethanolin 100 mM Tris-HCl buffer (pH 8.2) at room temperature. In a preparatoryexperiment for deciding the concentration of 2-Merchaptoethanol whichwas necessary to reduce cysteine residues of B3(Fab)-ext-PE38, it wasfound that the full reduction of cysteine residues was achived by adding20˜40 mM of 2-Merchaptoethanol. After the reduction, reduced proteincomplex was washed 1 time with washing buffer solution including 100 mMof MOPS (pH6.5) and washed 3 times more with 100 mM of TrisHCl (pH8.2).The washed protein complex was oxidized with oxidation buffer solutionincluding 5 mM of GSSG and 100 mM of TrisHCl (pH8.2) and warmed at 37°C. for 2 hrs. After the oxidation, 2×SDS sample buffer solution wasadded, the product was analyzed and stained with SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and Coomassie. The production rate of[B3(Fab)-ext-PE38]2 was analyzed through densitometry analysis that canmeasure the amount of protein on the SDS-PAGE samples. Using the datafrom SDS-PAGE, the yield of dimers in comparison with the total monomersof [B3(Fab)-ext-PE38]2 was calculated with the band intensity of[B3(Fab)-ext-PE38]2 divided by total band intensity which was the sum ofthe intensity of [B3(Fab)-ext-PE38]2 and B3(Fab)-ext-PE38 (See Table2.). B3(Fab)-ext-PE38 only and B3(Fab)-ext-PE38 bound to TR1 were usedas controls.

TABLE 2 The yield of [B3(Fab)-ext-PE38] 2 Antibody-toxin complexed withTR chains The yield (%) B3(Fab)-ext-PE38 N/A B3(Fab)-ext-PE38: TR1 N/AB3(Fab)-ext-PE38: TR2 N/A B3(Fab)-ext-PE38: TR3 47% ± 0.25B3(Fab)-ext-PE38: TR4 44% ± 0.19 B3(Fab)-ext-PE38: TR5 48% ± 0.12B3(Fab)-ext-PE38: TR6 36% ± 0.02 B3(Fab)-ext-PE38: TR7 52% ± 0.11

In the above experiment result, [B3(Fab)-ext-PE38]2 was not detected in3 samples (i.e., B3(Fab)-ext-PE38 only, complex with TR1 and complexwith TR2). Significant amount of inter-monomeric disulfide bond bridgeddimers were produced by the interaction of monomers in the complexeswith TR3˜TR7 chains. In samples of TR3˜TR7 complexes, the yield of[B3(Fab)-ext-PE38]2 was 47%, 44%, 48%, 36% and 52%, respectively.[B3(Fab)-ext-PE38]2 was produced within 2 hrs after the addition ofoxidizing agent and no significant increase was observed for extendedincubation. It was thus confirmed that cysteine residues in ext sequenceof B3(Fab)-ext-PE38 molecule have easier and closer contact to eachother via repeat-chains when they are bound to TR chains proteins.

Example 5 Construction of Protein G IgG-Binding Domain III Repeat-ChainGR8GR20, and GR2-2, -3, -4

We construct the plasmids that has two domain III of protein G with G4Slinker up to four times between the two domain III. The DNA sequence forthe domain III was obtained from chromosomal DNA of Streptococcus (KCTC3098) received from the Korean Collection for Type Cultures (KCTC), Theplasmid that has the domain III sequence, pGR1(Y. Lee et al, EnhancedFormation of Disulfide-bridged Dimer (Fab-PE38)2 Utilizing Repeats ofthe Fab Binding Domain of Protein G (2010) J. Biol. Chem. 285,5127-5131), was used for the site-directed metagenesis to constructpGR1-A that has an additional AgeI restriction site at the end of thecoding region of domain III of protein G and at the beginning of G4Ssequences.

Both primer 3 [5′-AGACCTTTAC GGTAACTCAA ACCGGTGGAG GCGGGTCCGG ATA-3′]and primer 4[5′-TATCCGGACC CGCCTCCACC GGTTTCAGTT ACCGTAAAGG TCT-3′] wereused for the Quick-change site-directed mutagenesis. After themutagenesis, the coding sequence of pGR1-A was confirmed by sequenceanalysis. The plasmid, pGR1-A, was digested by NdeI and BspEI. The smallfragment was purified. Also, pGR1 was digested by NdeI and AgeI, wherethe AgeI site is located after 6H is tag. The plasmid, pGR2-A, wasconstructed by ligation the large fragment of pGR1 that was produced bythe digestion of NdeI and AgeI to the small fragment of pGR1-A producedby the digestion of NdeI and BspEI. The digestion of pGR2-A by NdeI andAgeI gives large fragment of plasmid that has the G4S linker in front ofdomain III. Ligation of this large fragment to the small NdeI and BspEIfragment of pGR1-A, resulted pGR2-2 that has two units of G4S betweenfirst and second of domain III of Protein G. Subsequently, the plasmidspGR2-3 and GR2-4 which have three and four units of G4S linker wereconstructed in the same way that was used for the construction ofpGR2-2.

The construction for GR8˜20 were done in the same way as in the paper ofpGR1 (Y. Lee et al, Enhanced Formation of Disulfide-bridged Dimer(Fab-PE38)2 Utilizing Repeats of the Fab Binding Domain of Protein G(2010) J. Biol. Chem. 285, 5127-5131), and the FIGS. 4A and 4B is theschematic diagrams of expression plasmids constructions of GR1˜10.

FIG. 5 shows the results of SDS-PAGE of purified repeat-chains of domainIII of Protein G.

Example 6 Size-Exclusion Chromatography Analysis of the Complexes FormedBetween Fab-Toxin Monomer and Protein G Domain III Repeat-ChainGR1˜GR10, GR2-2, -3, -4, and the Disulfide-Bridged Dimer Formation inthe Complex

The immunoglobulin binding domain III of protein G binds both Fc and Fabfragments of IgG. It was reported that the immunoglobulin-binding region(domain III) of protein G can bind to the CH1 of the Fab fragment.Domain III of protein G binds to Fab through an antiparallel alignmentof the second β-strand from domain III with the seventh p-strand of CH1domain of Fab. The β/β interaction between two proteins accounts forfive hydrogen bonds between the CH1 domain of Fab fragment and domainIII of protein G. A further three hydrogen bounds also involve mainatoms from the CH1 domain. There do not appear to be any large changesin conformation of either domain III, or the CH1 domain on formation ofthe complex between the two proteins.

We construct two different types of the domain III repeat-chain. Firsttype of the constructs has one G4S amino acid between domain IIIs. Therepeat-chain that has up to 10 repeats of domain III was used toassociate with Fab-PE38 monomer. These repeat-chains are GR1 to GR10. Itis expected that multiple number of monomer on the repeat-chain, such asdimer, trimer or tetramer, are formed by association with domain IIIrepeat-chains.

Second, we also construct the proteins that have only two domain IIIwith different length of G4S linkers. It is made to see the differencesof complex formation of Fab-PE38 monomer with domain III repeat-chaindepending on the length of linker. These repeat-chain are GR2-2, GR2-3,and GR2-4, each having two, three, and four G4S linkers between twodomain III. These repeat-chain shows the effect of the length of linkerfor the formation of the complex of two Fab-PE38 with repeat-chaingiving dimeric form of two Fab-PE38 monomers. Using this construct ofGR2-2 to GR2-4, non-covalently associated complex could be obtainedeasily, and complex of dimer of Fab-PE38 monomer with the repeat-chainwas confirmed by size exclusion column.

FIG. 6 shows the results of size-exclusion chromatography of the GRcomplexes. The size-exclusion chromatography was performed withSuperdex-200TM HR column. The association of both Fab-ext-PE38 and GRproteins was done with different ratio. 715 μg of Fab-ext-PE38 wasassociated with each GR protein. From GR1˜GR3, Fab-ext-PE38 wasassociated with GR proteins as a molar ratio of 2:1. From GR4˜10, 715 μgof Fab-ext-PE38 was associated with 28 μg of each GR protein. All thechromatogram was aligned and stacked according to the same elutionvolume. The reference chromatograms of purified [Fab-ext-PE38]2 (D,dimer) and purified Fab-ext-PE38 (M, monomer) eluted with the samecondition.

FIG. 7 shows the results of size-exclusion chromatography of thecomplexes with GR2-2, -3, -4.

FIG. 8 shows the results of analysis on the mixture of [Fab-ext-PE38]2and Fab-ext-PE38 complexed with GR2 or GR3.

FIG. 9 shows the results of comparison of size-exclusion chromatogramsof mixtures of Fab-PE38 monomer and GR2-2, -3, -4 proteins.

FIG. 10 shows the results of size-exclusion chromatography for thepurification of the complexes of GR constructs and Fab-PE38 monomer.Proteins were finally purified by size-exclusion chromatography. All thechromatogram was the trace of UV absorption at 280 nm. The chromatogramswere aligned according to the same elution volume and stacked with thesame interval.

FIG. 11 shows the results of disulfide-bridged-dimer formation ofFab-toxin monomer by redox shuffling in the complex of Fab-toxin monomerwith GR10, GR2-2, GR2-3 and GR2-4. The products of the reaction wereanalyzed on 8% non-reducing SDS-PAGE.

TABLE 3 The calculated fraction of disulfide-bridged dimer formed byredox shuffle reaction of Fab-toxin monomer. Antibody-toxin bound tometal action of disulfide chelating bead dimer B3(Fab)-ext-PE38] n.d.*B3(Fab)-ext-PE38]:GR1 n.d.* B3(Fab)-ext-PE38]:GR2 n.d.*B3(Fab)-ext-PE38]:GR3 0.7 B3(Fab)-ext-PE38]:GR4 0.5B3(Fab)-ext-PE38]:GR5 0.6 B3(Fab)-ext-PE38]:GR6 0.3B3(Fab)-ext-PE38]:GR7 0.6 B3(Fab)-ext-PE38]:GR8 0.1B3(Fab)-ext-PE38]:GR9 0.1 B3(Fab)-ext-PE38]:GR10 0.3B3(Fab)-ext-PE38]:GR2-2 0.2 B3(Fab)-ext-PE38]:GR2-3 0.4B3(Fab)-ext-PE38]:GR2-4 0.3 *n.d.: not determined.

Example 7 Antibody Signal Effect Amplification by Protein G Domain IIIRepeat-Chain GR10 in Western Blot Analysis

Material and Method

Western blotting protocol from cell signaling Technology® was used withmodification, and modified version of direct ELISA protocol from Abcom®was used.

A. Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

1) 1×SDS Sample Buffer: 62.5 mM Tris-HCl (pH 6.8 at 25° C.), 2% w/v SDS,10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue or phenol red.

2) Transfer Buffer: 25 mM Tris base, 0.2 M glycine.

3) 10× Tris Buffered Saline (TBS): To prepare 1 liter of 10×TBS: 24.2 gTris base, 80 g NaCl; adjust pH to 7.6 with HCl (use at 1×).

4) Chicken egg albumin: (weight to volume [w/v]).

5) Blocking Buffer: 1×TBS, 0.1% Tween-20 with 2% w/v chicken serumalbumin.

6) Wash Buffer: 1×TBS, 0.1% Tween-20 (TBS/T).

7) Primary Antibody: mouse anti?beta-actin antibody from Santacruzbiotech.

8) Primary Antibody Dilution Buffer: 1×TBS, 0.1% Tween-20 with 2% w/vchicken serum albumin as indicated

9) Secondary Antibody: Goat anti-mouse beta actin-HRP.

10) Blotting Membrane: Nitrocellulose membranes (Wattman), PVDFmembranes (PALL).

11) GR recombinant protein. GR10

12) Luminol solution: 100 mM Tris/HCl pH 8.8, 1.25 mM luminol, 2 mM41PBA, 5.3 mM hydrogenperoxide

13) Super signal femto maximum sensitivity reagent (thermo scientific)

B. Protein Blotting

I. Sample preparation.

1) A431 or AGS carcinoma cell line was used for preparing the celllysate.

2) Aspirate media from cultures; wash cells with 1×PBS; aspirate.

3) Lyse cells by adding 1×SDS sample buffer.

4) Heat samples to 95-100° C. for 5 minutes.

5) Microcentrifuge for 5 minutes.

6) Load onto SDS-PAGE gel (10 cm×10 cm).

7) Electrotransfer to nitrocellulose or PVDF membrane.

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm×10 cm (100 cm2) of membrane; for differentsized membranes, adjust volumes accordingly.

I. Membrane Blocking

1) After transfer, wash nitrocellulose or PVDF membrane with appropriatevolume of TBS for 5 minutes at room temperature.

2) Incubate membrane in appropriate volume of blocking buffer for onehour at room temperature.

3) Wash three times for 5 minutes each with TBS/T.

II. Super-Complex Preparation.

1) Prepare and incubate GR10 and primary antibody (at the molar ratio asindicated in result) for 1 hr at 37° C.

2) Store on ice and keep the super-complex at 4° C. until use.

III. Primary Antibody Incubation

1) Incubate membrane and primary antibody (at the dilution as indicatedin result) in 10 ml primary antibody dilution buffer with gentleagitation for 1 hr at room temperature.

2) Wash three times for 5 minutes each with 15 ml of TBS/T.

3) Incubate membrane with the species appropriate HRP-conjugatedsecondary antibody (1:2000) in appropriate volume of blocking bufferwith gentle agitation for one hour at room temperature.

4) Wash three times for 5 minutes each with 15 ml of TBS/T.

5) Proceed to detection step in section D.

D. Detection of Proteins

1) Incubate membrane with luminol solution prepared or super signalfemto maximum sensitivity reagent (thermo scientific) with gentleagitation at room temperature.

2) Drain membrane of excess developing solution (do not let dry), wrapin plastic wrap and expose to x-ray film.

FIG. 12 shows the result of the amplification of chemiluminescencesignal by GR10 repeat-chain with conventional Western blotting reagents.

In FIG. 13-a, we observed that the super-complex gave approximately17-fold higher signal by the complex of monoclonal anti-β-actin mouseantibody and GR10. The complex formation of GR10 and primary antibodywas preformed according to the molar ratio, which is indicated inparenthesis. The primary antibody was used at the concentration of1:1000 dilution, and the super-complex was made at the sameconcentration of the primary antibody. For secondary antibody, goatanti-mouse-HRP conjugate was used for both experiments. Primary andsecondary antibody were incubated for an hour at RT. All the cell lysatesamples of the experiment was separated in the same 10% denaturingSDS-PAGE and transferred to nitrocellulose membrane, which is cut intothree pieces and probed with primary antibody. Highly sensitive ThermoSupersignal Femto substrate was used.

In FIG. 13-b, A431 clear lysate was separated by 10% denaturing SDS-PAGEand transferred to PVDF membrane to check whether the super-complexshows signal amplification that is comparable to that of nitrocellulosemembrane. The membrane was probed by primary antibody alone andsecondary antibody with conventional ECL, and instantly washed by TBSTand reprobed with Supersignal Femto substrate. We observed the increaseof signal and also huge background noise, which is due to nonspecificadsorption of secondary antibody on PVDF membrane and highly sensitiveSupersignal Femto substrate. Subsequently, the membrane was stripped bySDS and 2-mercaptoethanol treatment. The stripped membrane was reprobedby the super-complex with conventional ECL. Super-complex increased thesensitivity about 15-fold higher than the conventional method, which issimilar increase observed in previous experiment (a) with nitrocellulosemembrane. For this experiment we used conventional medium sensitivitychemiluminescence substrate. ECL was prepared by the method of Haan andBehrmann (2007). We could see similar signal amplification as seen onnitrocellulose membrane, and the amplified signal was compatible to thatof high sensitivity Supersignal Femto substrate but without backgroundnoise increase. Because of higher performance of PVDF membrane, lowamount of antigen could be detected with medium sensitivity substrate.

Example 8 Amplification of Antibody Signal by Protein G Domain IIIRepeat-Chain GR10 in Enzyme-Linked Immunosorbent Assay

Indirect ELISA

A. Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

1) Bicarbonate/carbonate coating buffer (100 mM) Antigen or antibodyshould be diluted in coating buffer to immobilize them to the wells:3.03 g Na2CO3, 6.0 g NaHCO3, 1000 ml distilled water pH 9.6.

2) PBS.

3) Blocking solution: 1% BSA, serum, in PBS.

4) Wash solution: PBS with detergent 0.05% (v/v) Tween20.

5) Antibody dilution buffer: Primary and secondary antibody should bediluted in 1× blocking solution to reduce Non specific binding.

6) Primary Antibody: mouse anti?beta-actin antibody from Santacruzbiotech.

7) Secondary Antibody: Goat anti-mouse beta actin-HRP.

8) TR recombinant protein. TR10

B. Coating Antigen to Microplate

1) A431 or AGS carcinoma cell line was used for preparing the celllysate.

2) Dilute the lysate to a final concentration of 20 μg/ml in carbonatecoating buffer.

3) Coat the wells of a microtiter plate with the diluted lysate bypipetting 50 μl of the lysate dilution in the top wells of the plate.

4) Cover the plate with lid and incubate 4° C. overnight.

5) Remove the coating solution and wash the plate twice by filling thewells with 200 μl PBS. The solutions or washes are removed by flickingthe plate over a sink. The remaining drops are removed by patting theplate on a paper towel.

C. Blocking

1) Block the remaining protein-binding sites in the coated wells byadding 200 μl blocking buffer, 1% BSA/PBS, per well.

2) Cover the plate with lid and incubate for at least 2 h at roomtemperature.

3) Wash the plate twice with PBS.

D. Incubation with the Antibody

1) Add 100 μl of the primary antibody or the super-complex, diluted atthe indicated concentration in blocking buffer immediately before use.

2) Cover the plate with a lid and incubate for 1 h at room temperature.

3) Wash the plate twice with PBS.

4) Add 100 μl of the secondary antibody-HRP, diluted at the optimalconcentration in blocking buffer immediately before use.

5) Cover the plate with a lid and incubate for 1 h at room temperature.

6) Wash the plate trice with PBS.

E. Detection

1) Dispense 100 μl of the TMB substrate solution per well with amultichannel pipet

2) After sufficient color development (30 min) add 100 μl of stopsolution to the wells.

3) Read the absorbance (optical density) of each well with a platereader.

FIG. 14 shows that GR10 significantly enhances the sensitivity of ELISA.In two different ELISA experiments, significant amplification of ELISAsignal was observed when the super-complex was used as a primaryantibody. AGS cell lysate was incubated overnight at 4° C. in regularcell culture tested 96 well plates and ELISA was done according to thestandard procedure. The super-complex was preformed according to themolar ratio, which is indicated in parenthesis. The primary antibody ismouse monoclonal anti-β-actin antibody. For secondary antibody, goatanti-mouse-HRP conjugate was used for both experiments. Primary andsecondary antibody were incubated for 1 hour at RT. The substrate, TMB,was incubated for 30 min at RT.

FIG. 15 shows that primary antibody and GR10 complex significantlyincrease the signal of ELISA and is rather consistent in the testedrange of secondary antibody dilution. In this ELISA experiments,significant amplification of ELISA signal was observed when thesuper-complex of monoclonal anti-β-actin antibody and GR10 was used as aprimary antibody. A431 cell lysate was used. The complex of GR10 andprimary antibody was made at the molar ratio of 1:10. For secondaryantibody, goat anti-mouse-HRP conjugate was used. 1 g of A431 celllysate was coated each well. The primary antibody was 10-fold seriallydiluted. With indicated primary antibody dilution factor, the secondaryantibody was 2-fold serially diluted.

Example 9 Sensitivity Enhancement of Influenza Rapid Antigen Test byProtein G Domain III Repeat-Chain GR10

The most common test kit using antibodies, influenza Rapid Antigen Testkits was used to see the amplification of the signal by GR10. Test kitsfrom SD BioLine (SD Inc.) or Green Cross was used. The antigen buffersolution, droppers, tubes, swabs and strips for clinical specimensamples are included in the kit. The antigen buffer was transferred totube filling the dropper to the indicated amount with the antigenbuffer. The swab samples of patients or the antigen sample solution wasadded to the tube and mixed more than 5 times. The GR10 protein wassimply added to the antigen solution in the tube together with theantigen and mixed. After removing the swab, the strip was inserted intothe tube, and the results were read after 10 to 15 minutes.

By simply adding GR10 to the commercially available rapid antigendiagnostic kits, the antigen band could be detected until the antigenwas 1000 fold more diluted compared the regular test. FIG. 16 shows thesignal amplification in rapid antigen test kit by GR10.

Example 10 The Use of GR Protein as a Labeling Agent to Antibody

Fluoro chromophore fluorescein isothiocyanate was conjugated to GR1(GR1-FITC) and GR1-FITC was used in immuno-fluorescence probing of thehuman squamous carcinoma A431 cell. The primary antibody was mouseanti-LC3 antibody, and the prepared GR1-FITC was used instead ofsecondary antibody. The cells were also probed with Rhodamine-phalloidin(sigma aldrich) for F-actin to compare, and observed under fluorescencemicroscope.

Very clear fluorescence image of the cell could be obtained with theGR1-FITC conjugate, even though the amount of the GR1-FITC protein usedwas a lot less than the case of secondary antibody-fluorescencechromophore conjugate. Like GR1-FITC, GR proteins can be linked,conjugated or fused to detection, signal or therapeutic functional groupto be used with antibodies, so that each antibody does not need to beconjugated with the functional group. GR proteins has small molecularweight and it is easy to produce and manipulate them. FIG. 17 showsImmuno-fluorescence probing of the human squamous carcinoma A431 cellwith GR1-FITC.

Example 11 Expression Plasmid Construction for Repeat-Chain Protein ofAntibody Binding Domain Domain B of Staphylococcus Aureus Protein A

The plasmid that contains the DNA sequence of Protein A Domain B wassynthesized (table 4) and obtained from the Bioneer Corporation. Theplasmid was put into Escherichia coli DH5a by transformation.

The plasmid was digested with NdeI and BspEI and the small DNA fragment(245 bp) was purified. The small fragment was cloned into the vectorfragment of pGR1 (pTR1) obtained by same enzyme digestion, and theplasmid pAR1 that contains one copy of domain B of protein A wasobtained. Protein A Domain B Nucleic acid sequence was checked bysequencing analysis of Macrogen corporation

The resulting plasmid, pAR1 (table 5) was digested with NdeI and BspEI.The small 245 bp fragment, encoding domain B and two of five amino acidGGGGS (G4S) sequence, was ligated with the large fragment of NdeI andAgeI digested pAR1. The resulting plasmid, pAR2, contains two repeat ofProtein A Domain B. pAR2 was digested with NdeI and AgeI again, and thelarge fragment was ligated with the 245 bp fragment to produce a newplasmid, pAR3, that has three time repeat of domain B. Using the samemethod, plasmids coding up to five repeats of protein A domain B wereconstructed.

TABLE 4 Nucleic acid sequence of Protein A Domain BNucleic acid sequence of Protein A Domain B (SEQ ID No: 3)5′-CATATGCATCATCATCATCACCACACCGGTTCTCAAGCCCCAAAAGCCGACAATAAATTTAATAAAGAGCAGCAGAACGCGTTTTATGAAATCTTGCATCTGCCGAATCTGAATGAAGAACAACGTAACGGATTCATTCAGAGCCTTAAAGATGATCCTAGTCAGTCCGCTAACTTACTCGCAGAAGCTAAGAAACTGAATGATGCACAGGCGCCGAAGGGAGGGGGTGGATCCGGTGGTGGCGGCTCCGGATAAGAATC-3′ *under line: coding sequence

TABLE 5 List of plasmids and proteins Plamid Repeat-chain protienReference pLR1~5 LR1~5: (His)₆-(B1-G₄S-G₄S)_(n), n = 1~5.^(a, b, c.)This specification ^(a)G4S: Amino acid sequence of GGGGS (SEQ ID No: 4);^(b)(His)6: six Histidine tag; and ^(c)B: Domain B of Staphylococcusaureus Protein A.

Repeat-chain proteins were overexpressed using previously describedmethod (J. H. Park, et al., Mol Cells 12 (2001) 398-402).

The crude cell lysate was subjected to Ni2+-chelating Sepharose fastflow chromatography (Amersham Bioscience, Sweden). The eluted proteinwas subjected to Hiload Superdex-75 pg or Hiload Superdex-200pg(26/60)(Amersham Bioscience, Sweden), and purified.

Example 12 Expression Plasmid Construction for Repeat-Chain Protein ofAntibody Binding Domain Domain B1 of Peptostreptococcus Magnus Protein L

The plasmid that contains the DNA sequence of Protein L Domain B1 wassynthesized (table 6) and obtained from the Bioneer Corporation. Theplasmid was put into Escherichia coli DH5a by transformation.

The plasmid was digested with NdeI and BspEI and the small DNA fragment(299 bp) was purified. The samll fragment was cloned into the vectorfragment of pGR1(pTR1) obtained by same enzyme digestion, and theplasmid pLR1 that contains one copy of domain B1 of protein L wasobtained. Protein L Domain B1 Nucleic acid sequence was checked bysequencing analysis of Macrogen corporation

The resulting plasmid, pLR1 (table 7) was digested with NdeI and BspEI.The small 299 bp fragment, encoding domain B1 and two of five amino acidGGGGS (G4S) sequence, was ligated with the large fragment of NdeI andAgeI digested pLR1. The resulting plasmid, pLR2, contains two repeat ofProtein L Domain B1. pLR2 was digested with NdeI and AgeI again, and thelarge fragment was ligated with the 299 bp fragment to produce a newplasmid, pLR3, that has three time repeat of domain B1. Using the samemethod, plasmids coding up to five repeats of protein L domain B1 (pLR5)were constructed.

TABLE 6 Nucleic acid sequence of Protein L Domain B1Nucleic acid sequence of Protein L Domain B1 (SEQ ID No: 5)5′-CATATGCATCACCATCACCATCATACCGGTATCAAGTTCGCCGGTAAAGAAGAAACGCCGGAAACCCCTGAGACAGACAGTGAAGAGGAAGTGACAATAAAAGCAAATCTGATTTTCGCCAACGGGTCAACCCAGACGGCCGAATTCAAAGGGACATTTGAAAAAGCAACTTCTGAGGCTTATGCATACGCGGACACTCTGAAGAAGGATAATGGTGAATATACCGTAGATGTTGCTGATAAAGGTTATACCCTGAATATTAAATTTGCGGGTGGCGGCGGCGGAAGCGGTGGCGGAGGTTCCGGATAAGAATTC-3′ *under line: coding sequence

TABLE 7 List of plasmids and proteins Plamid Repeat-chain protienReference pLR1~5 LR1~5: (His)₆-(B1-G₄S-G₄S)_(n), n = 1~5.^(a, b, c.)This specification ^(a)G4S: Amino acid sequence of GGGGS (SEQ ID No: 4);^(b)(His)6: six Histidine tag; and ^(c)B1: Domain B1 ofPeptostreptococcus magnus Protein L.

Repeat-chain proteins were overexpressed using previously describedmethod (J. H. Park, et al., Mol Cells 12 (2001) 398-402).

The crude cell lysate was subjected to Ni2+-chelating Sepharose fastflow chromatography (Amersham Bioscience, Sweden). The eluted proteinwas subjected to Hiload Superdex-75 pg or Hiload Superdex-200 pg (26/60)(Amersham Bioscience, Sweden), and purified.

Example 13 Expression Plasmid Construction for Repeat-Chain Protein ofAntibody Binding Domain Domain B1 of Peptostreptococcus Magnus Protein Land Antibody Binding Domain Domain B of Staphylococcus aureus Protein A

The plasmid that contains the DNA sequence of Protein L Domain B1 wassynthesized and used to construct pLR1.

The plasmid pLR1 was digested with NdeI and BspEI and the small DNAfragment (299 bp) was purified. The samll fragment was cloned into thevector fragment of pAR1 obtained by same enzyme digestion, and theplasmid pLAR1 that contains one copy of domain B1 of protein L connectedto one copy of domain B of protein A (B1-B) was obtained. The nucleicacid sequence was checked by sequencing analysis of Macrogencorporation.

The resulting plasmid, pLAR1 (table 8) was digested with NdeI and BspEI.The small 521 bp fragment, encoding domain B1-B and two of five aminoacid GGGGS (G4S) sequence between domains, was ligated with the largefragment of NdeI and AgeI digested pLAR1. The resulting plasmid, pLAR2,contains two repeat of B1-B. pLAR2 was digested with NdeI and AgeIagain, and the large fragment was ligated with the 521 bp fragment toproduce a new plasmid, pLAR3, that has three time repeat of domain B1-B.Using the same method, plasmids coding up to three repeats of protein Ldomain B1 and protein A domain B (B1-B) (pLAR3) were constructed.

TABLE 8 Nucleic acid sequence of Domain B1-Domain BNucleic acid sequence of Domain B1-Domain B (SEQ ID No: 6)5′-CATATGCATCACCATCACCATCATACCGGTATCAAGTTCGCCGGTAAAGAAGAAACGCCGGAAACCCCTGAGACAGACAGTGAAGAGGAAGTGACAATAAAAGCAAATCTGATTTTCGCCAACGGGTCAACCCAGACGGCCGAATTCAAAGGGACATTTGAAAAAGCAACTTCTGAGGCTTATGCATACGCGGACACTCTGAAGAAGGATAATGGTGAATATACCGTAGATGTTGCTGATAAAGGTTATACCCTGAATATTAAATTTGCGGGTGGCGGCGGCGGAAGCGGTGGCGGAGGTTCCGGTTCTCAAGCCCCAAAAGCCGACAATAAATTTAATAAAGAGCAGCAGAACGCGTTTTATGAAATCTTGCATCTGCCGAATCTGAATGAAGAACAACGTAACGGATTCATTCAGAGCCTTAAAGATGATCCTAGTCAGTCCGCTAACTTACTCGCAGAAGCTAAGAAACTGAATGATGCACAGGCGCCGAAGGGAGGGGGTGGATCCGGTGGTGGCGGCTCCGGATAAGAATTC-3′ *under line: coding sequence

TABLE 9 List of plasmids and proteins Plamid Repeat-chain protienReference pLAR1~3 LAR1~3: (His)₆-(B1-G₄S-G₄S-B-G₄S-G₄S)_(n), This n =1~3.^(a,b,c,d) specification ^(a)G4: Amino acid sequence of GGGG (SEQ IDNo: 7); ^(b)(His)6: six Histidine tag; ^(c)B1: Peptostreptococcus magnusProtein L Domain B1; and ^(d)B: Staphylococcal aureus Protein A DomainB.

Repeat-chain proteins were overexpressed using previously describedmethod (J. H. Park, et al., Mol Cells 12 (2001) 398-402).

The crude lysate was subjected to Ni2+-chelating Sepharose fast flowchromatography (Amersham Bioscience, Sweden). The eluted protein wassubjected to Hiload Superdex-75 pg or Hiload Superdex-200 pg(26/60)(Amersham Bioscience, Sweden), and purified.

Example 14 Cross Binding of Gold Antibody/Repeat-Chain Complex orSuper-Complex to Test Line Antibody of Rapid Antigen Test Kit

The cross-bindings of the gold antibody/repeat-chain complexes orsuper-complexes to the detection line antibody on influenza rapidantigen test kit strip were tested without adding virus antigens. Thiscross-binding without the antigen is brought by the binding of emptyimmunoglobulin binding domain of the repeat-chain in therepeat-chain/gold antibody complex or super-complexes to the antibodiesof the test line fixed on the strip. We used SD influenza A/B rapid testkit and Green cross influenza A/B rapid test kit. They consist of teststrip, assay buffer, disposable droppers and disposable swab. Assaybuffer was transferred into a test tube, and the GR, AR, LR, LAR seriesproteins were added into the test tube. The tube was swirled at leastfive times to mix. The test strip was placed into the test tube with thearrows mark on the test strip pointing down and read result after 10˜15min.

The cross binding was detected by GR4, GR5, GR7GR10, GR15GR20. Theprotein amounts of the repeat-chain used were 10 μg, 1 μg and 0.1 μg forSD strips. The bands of Green cross strips were clearer than SD strips.There was no band detected at 0.1 μg of repeat-chain (FIG. 18). AR5,LR3, LR5, LAR1, LAR2, LAR3 also gave cross binding band on SD strip. ButGreen Cross strip did not give any band with LR series protein. ProteinL binds only to kappa light chain and it indicated that the antibodyused on Green Cross strip was not kappa light chain. All of AR, LR, LARproteins did not give cross binding bands at below 0.1 μg (FIG. 19).

Example 15 The Signal Amplification by Repeat-Chains on Influenza RapidAntigen Test Kit

Rapid Antigen Test is the typical diagnosis technique using antibody asdetection probe. The amplifications of the signal effect of InfluenzaRapid Antigen Test Kit by repeat chain GR, AR, LR and LAR seriesrepeat-chains were tested. We used SD influenza A/B rapid test kit andGreen cross influenza A/B rapid test kit. We used same procedure aspreviously described example. The amounts of GR, AR, LR, LAR proteinsused are indicated in each figure. The virus antigen samples were addedin 10-fold serial dilutions.

For GR5, the antigen band could be detected until the antigen was 10̂−8fold diluted. (FIG. 20)

For GR10, the antigen band could be detected until the antigen was 10̂−8fold diluted. (FIG. 21)

For GR15, the antigen band could be detected until the antigen was 10̂−9fold diluted. (FIG. 22)

For GR20, the antigen band could be detected until the antigen was 10̂−9fold diluted. (FIG. 23)

For AR5, there was no amplification of the signal detected. (FIG. 24)

For LR5, the antigen band could be detected until the antigen was 10̂−6fold diluted. (FIG. 25)

For LAR3, the antigen band could be detected until the antigen was 10̂−8fold diluted. (FIG. 26)

Example 16 Observation of Supercomplex Precipitation BetweenRepeat-Chain GR, AR, LR, LAR and IgG

IgG solution was made by solving IgG powder of Equitech Bio, in PBS. TheIgG solution was placed on rocker for 2 hours, and then powder whichwasn't solved completely in the solution was removed by centrifugationfor 21000 rpm, 2 hours at 4° C. GR1˜20 and IgG solutions were used afterquantitative analysis by Bicinchoninic Acid protein assay, and dilutedto proper concentrations. The GR1˜20 solutions are mixed with IgGsolution in micro-centrifuge tubes and incubated at room temperatureovernight. After the incubation, the precipitation was collected bycentrifuge at 13000 rpm for 30 minutes at 20° C. If precipitation wasformed, it could be observed with bare eyes at this point. Supernatantwas removed and placed a clear new tube by using micro-pipets, and then500 μl of 75% ethanol was added to the tube and removed to wash outremaining solutions which could be on the inner surface of the tube.After evaporating remaining ethanol completely the pellet was put onSDS-PAGE. The results showed that when IgG was mixed with GR10 in molarratios of 1:1, 5:1 and 10:1, one in 5:1 ratio had more precipitationthan others (FIG. 27).

IgG and GR10 were mixed in 5:1 molar ratio, and same amounts of GR1 & 2in gram were mixed with IgG and incubated. More precipitation and pelletprotein bands on a SDS-PAGE gel were observed with GR10 than GR1 or GR2(FIG. 28). When GR proteins exist in same gram amounts, the totalamounts of binding domains are the same, but in GR10, the ten domainsare linked to form a single repeat-chain. When the domains are linked toform a longer repeat-chain, it is able to form super-complex muchbetter.

IgG and GR10 were mixed in 5:1 molar ratio, and same amounts of GR1˜9 ingram were mixed with IgG and incubated. The final concentration of IgGwas 400 μg/ml. Precipitation which could be observed with bare eyes(FIG. 29) and pellet IgG bands on a SDS-PAGE gel (FIG. 30) could be seenfrom GR3 to GR10. This results showed that the precipitatiblesuper-complex by cross-linking between complexes can be formed from GR3that has three D(III) domains and one GGGGS linker between the domainsin it.

IgG only control sample and BSA in place of GR control sample did notgive precipitation. This indicated that precipitation did not formedbecause the addition of other protein in the solution caused thedecrease of solubility of IgG protein, but precipitations were formed bythe addition of the repeat chain which can cross-bind the complexesforming super-complex of heavy molecular weight.

To see whether GRs bigger than GR10 can make super-complex with IgG, IgGand GR10 were mixed in 5:1 ratio, and GR1, 3, 5, 15 and 20 were testedin the same way as above. Precipitation was observed in all samplesexcept IgG+GR1 and IgG only samples. The SDS-PAGE results showedprecipitation were formed from GR3 to GR20 (FIG. 31). GRs bigger thanGR10 like GR15 or 20 can also form precipitatible super-complex.

When the final concentrations of IgG were decreased serially and testedin the same way, there were no precipitations below 25 μg/ml of IgG.These results indicated that precipitatible super-complex formation ofGR with IgG was dependent on concentration of IgG.

Using the same procedure, AR1, 3, 5, LR1, 3, 5 and LAR1, 2, 3 wereincubated with IgG. The concentration of IgG of 500 μg/ml was used.Precipitations couldn't be observed for AR, LR and LAR repeat-chainproteins. These repeat-chain proteins could not make precipitatibleinsoluble super-complex.

Example 17 The Increase of Antibody Signal in Enzyme-LinkedImmunosorbent Assay (ELISA) by GR10, GR20, AR5, LR5, LAR3

The procedures used are same as in the example 16.

The signal intensities were increased by GR10, GR20, LR5, LAR3, but notby AR5(Table 10).

TABLE 10 The increase of antibody signal in enzyme-linked immunosorbentassay (ELISA) by GR10, GR20, AR5, LR5, LAR3. Sample A450 nm value FoldIncrease IgG + (No repeat 0.79 1.0 chain) IgG + GR10 2.49 3.2 IgG + GR202.43 3.1 IgG + AR5 0.89 1.1 IgG + LR5 1.07 1.4 IgG + LAR3 1.09 1.4

INDUSTRIAL APPLICABILITY

The present invention relates to formation of multimers formed by bondbridges. With repeat-chains of affinity domains having specific bindingaffinity, the formation of a repeat-chain/multiple-monomer complex canbe achieved, and thus a formed repeat-chain/multiple-monomer complex canbe used to produce multimers linked by bond bridges. Remarkably highformation rate is provided by a method of the present invention andleads to formation of dimers linking with bond bridges in mass quantity;therefore, the present invention is industrially applicable.

Monomers can also be made with many kinds of substances includingligand, or a part fragment of the ligand having binding affinity such asTGF alpha, TGF beta, IL2, IL6, TNF or GMSCF, and various kinds of ligandreceptors or a part fragment of ligand receptors having binding affinitysuch as TBP1, TBP2, IFN alpha or beta, or gonadotropin receptors.(Nienhaus, G. Ulrich. Protein-ligand interactions: methods andapplications. Humana Press, 2005).

In addition, The followings may be used to form monomers: various kindsof enzymes catalyzing prodrugs transformation or detection,decomposition and formation of substance, proteins including functionalgroup of cytotoxic toxin, organisms such as virus for gene therapy,cation tail compounds for DNA delivery, liposome produced by chemicalengineering method for delivery of drug, biosensor for real-timedetection of target molecule, or prodrug, or other functional groups[References: (Farah, R. A., et al., Crit. Rev. Eukaryot. Gene Expr. 8,321-356, 1998) (Trail, P. A., et al., Science 261, 212-215,1993)(Hinman, L. M., et al., Cancer Res. 53, 3336-3342, 1993)(Pastan, I.Biochim. Biophys. Acta 1333, C1-C6, 1997)(Kreitman, P. J., et al., J.Clin. Oncol. 18, 1622-1636, 2000)(Zalutsky, M. R. & Vaidyanathan, G.Curr. Pharm. Des. 6, 1433-1455, 2000)(Goldenberg, D. M. in Clinical Usesof Antibodies (eds Baum, R. P., et al.)1-13(Kluwer academic, TheNetherlands, 1991)) (Lode, H. N. & Reisfeld, R. A. Immunol. Res. 21,279-288, 2000)(Penichet, M. L. & Morrison, S. L. J. Immunol. Methods248, 91-101, 2001) (Lasic, D. D. & Papahadjopoulos, D. Science 267,1275-1276, 1995) (Park. J. W., et al., Proc. Natl. Acad. Sci. USA 92,1327-1331, 1995)(Niculescu-Davaz, I., et al., Anticancer Drug Des. 14,517-538, 1999)(SToldt, H. S., et al., Eur. J. Cancer 33, 186-192,1997)].

In nature, there are various kinds of substances having binding affinityrelationship to each other. Ligands and acceptors, antibody and antigen,homodimer, heterodimer, or proteins forming multimers are the knownsubstances. By using these substances having inter-binding-affinity asmonomers and affinity domains, multimers linked with bond bridges may beprepared in large quantity from monomers based on a method of thepresent invention.

In the present invention, the repeat-chains having specific bindingaffinity are characterized to form a repeat-chain/multiple-monomercomplex. Therefore, as fixing the repeat-chains on resin, it is possibleto perform affinity purification with high efficiency for thepurification of useful protein molecules in monomeric or multimeric formin biotechnology and medical industry (Zachariou M., AffinityChromatography: Methods and Protocols (Methods in Molecular Biology)Humana Press; 2nd edition).

The present invention relates to a super-complex prepared bycross-binding between repeat-chain/monomer complexes, and a method foramplifying the effect of monomer through the formation of the saidsuper-complex. Particularly, the repeat-chain/monomer complex isprepared by containing repeat-chains of binding domain having bindingspecificity to monomers as active ingredients, and then thesuper-complex is prepared by cross-binding between such complexes. Sincethe super-complex contains multiple monomers, the biological andchemical effect becomes very strong, based on which the presentinvention provides a method for amplifying the effect of monomerincluding signal amplification, etc.

In this invention, multiple monomers and repeat-chains are bound to eachother to form a complex. By cross-binding between such complexes, aninsoluble super-complex can be generated at high concentration, whichgives precipitations. Aggregation, precipitation, and size of thesuper-complex depend on the structures of monomer and repeat-chain. Thenumber of repeat in the binding domain affects the cross-binding betweencomplexes. Water-solubility and molecular size of the monomer affect thechance of cross-binding. The said super-complex demonstrates multiplebiological and chemical effect on target of the monomer since itcontains multiple monomers therein. Therefore, it can be effectivelyused for the amplifications of detection signal, reaction effect, andtherapeutic treatment effect, etc.

1.-15. (canceled)
 16. A method for preparing a super-complex, whichcomprises the following steps: 1) preparing repeat-chains which containa single or multiple kinds of monomer-specific binding domains having atleast two binding sites for a monomer repeated therein; 2) preparingrepeat-chain/multiple-monomer complexes by mixing the repeat-chains ofstep 1) and the monomers having at least two binding sites for therepeat-chains; and 3) generating sugar-complex aggregates of thecomplexes by forming cross-binding between therepeat-chain/multiple-monomer complexes of step 2).
 17. The method asset forth in claim 16, wherein the monomer is a protein.
 18. The methodas set forth in claim 16, wherein the monomer is selected from the groupconsisting of antibodies, ligands, receptors or fragments thereof, orrecombinants thereof, or derivatives thereof, or fusions of biologicalor chemical functional group therewith.
 19. The method as set forth inclaim 18, wherein the antibody is selected from the group consisting offragments of an antibody, Fab fragment, fragments containing Fabfragment, Fv fragment, fragments containing Fv fragment, Fc fragment,and fragments containing Fc fragment.
 20. The method as set forth inclaim 16, wherein the binding domain is a protein.
 21. (canceled) 22.The method as set forth in claim 16, wherein the binding domain isselected from the group consisting of streptococcal protein G,Staphylococcus aureus protein A, Peptostreptococcus magnus protein L,and their derivatives.
 23. (canceled)
 24. (canceled)
 25. A super-complexprepared by the method of claim
 16. 26. A method for amplifying theeffect of monomer, which comprises the step of preparing a super-complexbound to the target of the monomer by mixing the super-complex of claim25 to the target of the monomer.
 27. The method for amplifying theeffect of monomer as set forth in claim 26, wherein the additional stepof measuring the effect of monomer on the target of the monomer isincluded.
 28. The method for amplifying the effect of monomer as setforth in claim 26, wherein the target of the monomer is selected fromthe group consisting of antigens, antibodies, peptides, proteins,bacteria, viruses, and fungi.
 29. The method for amplifying the effectof monomer as set forth in claim 28, wherein the bacteria are selectedfrom the group consisting of Helicobacter pylori, Mycobacteriumtuberculosis, and Chlamydia trachomatis.
 30. The method for amplifyingthe effect of monomer as set forth in claim 28, wherein the virus isselected from the group consisting of influenza, foot and mouth diseasevirus, human papilloma virus (HPV), Dengue fever virus, hepatitis Cvirus, and hepatitis B surface antigen and antibody.
 31. The method foramplifying the effect of monomer as set forth in claim 27, wherein themeasurement of the effect of monomer is performed by using secondaryprobe(antibody)-marker conjugate and biological and chemical labelingfunction of the marker.
 32. The method for amplifying the effect ofmonomer as set forth in claim 31, wherein the marker is selected fromthe group consisting of horseradish peroxidase (HRP), alkalinephosphatase, colloid gold, fluorescein, Quantum dot, glucose oxidase,luciferase, beta-D-galactosidase, malate dehydrogenase (MDH),acetylcholinesterase, radio-isotope, and dye.
 33. The method foramplifying the effect of monomer as set forth in claim 31, wherein thechromogenic substrate is selected from the group consisting of3,3′,5,5′-tetramethyl bezidine (TMB),2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), diaminobenzidine (DAB),3-amino-9-ethylcarbasole, 5-bromo-4-chloro-3-indolylphosphate/iodonitrotetrazolium (BCIP/INT), new fuchin (NF), and fast redTR salts. 34.-37. (canceled)
 38. A method for preparing a complex withmultiple-monomer/repeat-chain-detection functional group, comprising thefollowing steps: 1) preparing a repeat-chain-detection functional groupby linking, conjugating, or fusing detection functional group torepeat-chains which contain a single or multiple kinds ofmonomer-specific binding domains having one or more binding sites for amonomer repeated therein; and 2) preparing repeat-chain-detectionfunctional group/multiple-monomer complexes by mixing therepeat-chain-detection functional group of step 1) and the monomershaving one or more binding sites for the repeat-chains.
 39. A method forpreparing a super-complex with multiple-monomer/repeat-chain-detectionfunctional group, which comprises the following steps: 1) preparingrepeat-chain-detection functional group by linking, conjugating, orfusing detection functional group to repeat-chains which contain asingle or multiple kinds of monomer-specific binding domains having atleast two binding sites for a monomer repeated therein; 2) preparingrepeat-chain-detection functional group/multiple-monomer complexes bymixing the repeat-chain-detection functional group of step 1) and themonomers having at least two binding sites for the repeat-chains; and 3)generating aggregates of the complexes by forming cross-binding betweenthe repeat-chain-detection functional group/multiple-monomer complexesof step 2).
 40. The method as set forth in claim 38 or claim 39, whereinthe detection functional group is selected from the group consisting ofCy-3, Cy-5, FITC, GFP (green fluorescent protein), RFP (red fluorescentprotein), and Texas Red, colloid gold, fluorescein, Quantum dot,radio-isotope, and dye. 41.-42. (canceled)
 43. A super-complex preparedby the method of claim
 39. 44. A method for detecting the target of themonomer, containing the step of forming a super-complex bound to thetarget of the monomer by mixing themultiple-monomer/repeat-chain-detection functional group super-complexof claim 43 to the target of the monomer.
 45. The method for detectingthe target of the monomer as set forth in claim 44, wherein the step ofmeasuring the detection level of monomer for the target of the monomeris additionally included.